Methods of using antibodies to VEGF-C

ABSTRACT

Provided are purified and isolated VEGF-C polypeptides capable of binding to at least one of KDR receptor tyrosine kinase (VEGFR-2) and Flt4 receptor tyrosine kinase (VEGFR-3); analogs of such peptides that have VEGF-C-like or VEGF-like biological activities or that are VEGF or VEGF-C inhibitors; polynucleotides encoding the polypeptides; vectors and host cells that embody the polynucleotides; pharmaceutical compositions and diagnostic reagents comprising the polypeptides; and methods of making and using the polypeptides. Methods of inhibiting endothelial cell proliferation are also provided.

This application is a continuation of U.S. patent application Ser. No.10/792,461 filed Mar. 3, 2004 (now U.S. Pat. No. 7,423,145),incorporated herein by reference in its entirety, which is acontinuation of U.S. patent application Ser. No. 09/534,376, filed Mar.24, 2000, now U.S. Pat. No. 6,818,220; which is a continuation of U.S.patent application Ser. No. 09/355,700, filed Nov. 5, 1999 now U.S. Pat.No. 6,361,946, which is a 35 USC §371 U.S. National Stage filing ofInternational Application No. PCT/US98/01973, filed Feb. 2, 1998, whichis a continuation-in-part of U.S. patent application Ser. No.08/795,430, filed Feb. 5, 1997, now U.S. Pat. No. 6,130,071. This patentapplication also is a continuation-in-part of International PatentApplication PCT/F196/00427, filed Aug. 1, 1996; which is acontinuation-in-part of U.S. patent application Ser. No. 08/671,573,filed Jun. 28, 1996, now U.S. Pat. No. 6,645,933; which is acontinuation-in-part of U.S. patent application Ser. No. 08/601,132,filed Feb. 14, 1996, now U.S. Pat. No. 6,403,088; which is acontinuation-in-part of U.S. patent application Ser. No. 08/585,895,filed Jan. 12, 1996, now U.S. Pat. No. 6,245,530; which is acontinuation-in-part of U.S. patent application Ser. No. 08/510,133,filed Aug. 1, 1995, now U.S. Pat. No. 6,221,839.

FIELD OF THE INVENTION

The present invention generally relates to the field of geneticengineering and more particularly to growth factors for endothelialcells and growth factor genes.

BACKGROUND OF THE INVENTION

Developmental growth, the remodeling and regeneration of adult tissues,as well as solid tumor growth, can only occur when accompanied by bloodvessel formation. Angioblasts and hematopoietic precursor cellsdifferentiate from the mesoderm and form the blood islands of the yolksac and the primary vascular system of the embryo. The development ofblood vessels from these early (in situ) differentiating endothelialcells is termed vasculogenesis. Major embryonic blood vessels arebelieved to arise via vasculogenesis, whereas the formation of the restof the vascular tree is thought to occur as a result of vascularsprouting from pre-existing vessels, a process called angiogenesis,Risau et al., Devel. Biol., 125:441-450 (1988).

Endothelial cells give rise to several types of functionally andmorphologically distinct vessels. When organs differentiate and begin toperform their specific functions, the phenotypic heterogeneity ofendothelial cells increases. Upon angiogenic stimulation, endothelialcells may re-enter the cell cycle, migrate, withdraw from the cell cycleand subsequently differentiate again to form new vessels that arefunctionally adapted to their tissue environment. Endothelial cellsundergoing angiogenesis degrade the underlying basement membrane andmigrate, forming capillary sprouts that project into the perivascularstroma. Ausprunk et al., Microvasc. Rev., 14:51-65 (1977). Angiogenesisduring tissue development and regeneration depends on the tightlycontrolled processes of endothelial cell proliferation, migration,differentiation, and survival. Dysfunction of the endothelial cellregulatory system is a key feature of many diseases. Most significantly,tumor growth and metastasis have been shown to be angiogenesisdependent. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992).

Key signals regulating cell growth and differentiation are mediated bypolypeptide growth factors and their transmembrane receptors, many ofwhich are tyrosine kinases. Autophosphorylated peptides within thetyrosine kinase insert and carboxyl-terminal sequences of activatedreceptors are commonly recognized by kinase substrates involved insignal transduction for the readjustment of gene expression inresponding cells. Several families of receptor tyrosine kinases havebeen characterized. Van der Geer et al., Ann. Rev. Cell Biol.,10:251-337 (1994). The major growth factors and receptors transducingangiogenic stimuli are schematically shown in FIG. 1.

Fibroblast growth factors are also known to be involved in theregulation of angiogenesis. They have been shown to be mitogenic andchemotactic for cultured endothelial cells. Fibroblast growth factorsalso stimulate the production of proteases, such as collagenases andplasminogen activators, and induce tube formation by endothelial cells.Saksela et al., Ann. Rev. Cell Biol., 4:93-126 (1988). There are twogeneral classes of fibroblast growth factors, FGF-1 and FGF-2, both ofwhich lack conventional signal peptides. Both types have an affinity forheparin, and FGF-2 is bound to heparin sulfate proteoglycans in thesubendothelial extracellular matrix from which it may be released afterinjury. Heparin potentiates the stimulation of endothelial cellproliferation by angiogenic FGFs, both by protecting againstdenaturation and degradation and dimerizing the FGFs. Culturedendothelial cells express the FGF-1 receptor but no significant levelsof other high-affinity fibroblast growth factor receptors.

Among other ligands for receptor tyrosine kinases, the platelet derivedgrowth factor, PDGF-BB, has been shown to be weakly angiogenic in thechick chorioallantoic membrane. Risau et al., Growth Factors, 7:261-266(1992). Transforming growth factor α (TGFα) is an angiogenic factorsecreted by several tumor cell types and by macrophages. Hepatocytegrowth factor (HGF), the ligand of the c-met proto-oncogene-encodedreceptor, also is strongly angiogenic.

Recent evidence shows that there are endothelial cell specific growthfactors and receptors that may be primarily responsible for thestimulation of endothelial cell growth, differentiation and certaindifferentiated functions. The best studied of these is vascularendothelial growth factor (VEGF), a member of the PDGF family. Vascularendothelial growth factor is a dimeric glycoprotein of disulfide-linked23 kD subunits. Other reported effects of VEGF include the mobilizationof intracellular calcium, the induction of plasminogen activator andplasminogen activator inhibitor-1 synthesis, stimulation of hexosetransport in endothelial cells, and promotion of monocyte migration invitro. Four VEGF isoforms, encoded by distinct mRNA splice variants,appear to be equally capable of stimulating mitogenesis in endothelialcells. However, each isoform has a different affinity for cell surfaceproteoglycans, which behave as low affinity receptors for VEGF. The 121and 165 amino acid isoforms of VEGF (VEGF121 and VEGF165) are secretedin a soluble form, whereas the isoforms of 189 and 206 amino acidresidues remain cell surface-associated and have a strong affinity forheparin. VEGF was originally purified from several sources on the basisof its mitogenic activity toward endothelial cells, and also by itsability to induce microvascular permeability, hence it is also calledvascular permeability factor (VPF).

Two high affinity receptors for VEGF have been characterized:VEGFR-1/Flt-1 (fins-like tyrosine kinase-1) and VEGFR-2/KDR/Flk-1(kinase insert domain containing receptor/fetal liver kinase-1). Thosereceptors are classified in the PDGF-receptor family, but they haveseven rather than five immunoglobulin-like loops in their extracellulardomain (see FIG. 1), and they possess a longer kinase insert thannormally observed in this family. The expression of VEGF receptorsoccurs mainly in vascular endothelial cells, although some may bepresent on hematopoietic progenitor cells, monocytes, and melanomacells. Only endothelial cells have been reported to proliferate inresponse to VEGF, and endothelial cells from different sources showdifferent responses. Thus, the signals mediated through VEGFR-1 andVEGFR-2 appear to be cell type specific. The VEGF-related placentagrowth factor (PlGF) was recently shown to bind to VEGFR-1 with highaffinity. PlGF was able to enhance the growth factor activity of VEGF,but it did not stimulate endothelial cells on its own. Naturallyoccurring VEGF/PlGF heterodimers were nearly as potent mitogens as VEGFhomodimers for endothelial cells. Cao et al., J. Biol. Chem.,271:3154-62 (1996).

The Flt4 receptor tyrosine kinase (VEGFR-3) is closely related instructure to the products of the VEGFR-1 and VEGFR-2 genes. Despite thissimilarity, the mature form of Flt4 differs from the VEGF receptors inthat it is proteolytically cleaved in the extracellular domain into twodisulfide-linked polypeptides. Pajusola et al., Cancer Res.,52:5738-5743 (1992). The 4.5 and 5.8 kb Flt4 mRNAs encode polypeptideswhich differ in their C-termini due to the use of alternative 3′ exons.Isoforms of VEGF or PlGF do not show high affinity binding to Flt4 orinduce its autophosphorylation.

Expression of Flt4 appears to be more restricted than the expression ofVEGFR-1 or VEGFR-2. The expression of Flt4 first becomes detectable byin situ hybridization in the angioblasts of head mesenchyme, thecardinal vein, and extraembryonically in the allantois of 8.5 day p.c.mouse embryos. In 12.5 day p.c. embryos, the Flt4 signal is observed indeveloping venous and presumptive lymphatic endothelia, but arterialendothelia appear negative. During later stages of development, Flt4mRNA becomes restricted to developing lymphatic vessels. The lymphaticendothelia and some high endothelial venules express Flt4 mRNA in adulthuman tissues and increased expression occurs in lymphatic sinuses inmetastatic lymph nodes and in lymphangioma. These results support thetheory of the venous origin of lymphatic vessels.

Five endothelial cell specific receptor tyrosine kinases, Flt-1(VEGFR-1), KDR/Flk-1 (VEGFR-2), Flt4 (VEGFR-3), Tie, and Tek/Tie-2 haveso far been described, which possess the intrinsic tyrosine kinaseactivity essential for signal transduction. Targeted mutationsinactivating Flt-1, Flk-1, Tie, and Tek in mouse embryos have indicatedtheir essential and specific roles in vasculogenesis and angiogenesis atthe molecular level. VEGFR-1 and VEGFR-2 bind VEGF with high affinity(K_(d) 16 pM and 760 pM, respectively) and VEGFR-1 also binds therelated placenta growth factor (PlGF; K_(d) about 200 pM). A ligand forTek is reported in PCT patent publication WO 96/11269.

SUMMARY OF THE INVENTION

The present invention provides a ligand, designated VEGF-C, for the Flt4receptor tyrosine kinase (VEGFR-3). Thus, the invention provides apurified and isolated polypeptide which is capable of binding to theFlt4 receptor tyrosine kinase. Preferably, an Flt4 ligand of theinvention is capable of stimulating tyrosine phosphorylation of Flt4receptor tyrosine kinase in a host cell expressing the Flt4 receptortyrosine kinase. Preferred ligands of the invention are mammalianpolypeptides. Highly preferred ligands are human polypeptides. Asexplained in detail below, dimers and multimers comprising polypeptidesof the invention linked to each other or to other polypeptides arespecifically contemplated as aspects of the invention.

In one embodiment, an Flt4 ligand polypeptide has a molecular weight ofapproximately 23 kD as determined by SDS-PAGE under reducing conditions.For example, the invention includes a ligand composed of one or morepolypeptides of approximately 23 kD which is purifyable from conditionedmedia from a PC-3 prostatic adenocarcinoma cell line, the cell linehaving ATCC Acc. No. CRL 1435. Amino acid sequencing of this PC-3cell-derived ligand polypeptide revealed that the ligand polypeptidecomprises an amino terminal amino acid sequence set forth in SEQ ID NO:5. The present invention also provides a new use for the PC-3 prostaticadenocarcinoma cell line which produces an Flt4 ligand. In a preferredembodiment, the ligand may be purified and isolated directly from thePC-3 cell culture medium.

In a highly preferred embodiment, the ligand polypeptide comprises afragment of the amino acid sequence shown in SEQ ID NO: 8 which bindswith high affinity to the human Flt4 receptor tyrosine kinase. It willbe understood that the term “high affinity,” in the context of apolypeptide ligand of a receptor tyrosine kinase, typically reflects abinding relationship characterized by sub-nanomolar dissociationconstants (K_(d)), as reported herein for VEGF-C binding to VEGFR-2 andVEGFR-3, and reported elsewhere in the art for the binding of VEGF,PlGF, PDGF, and other factors to their receptors. Exemplary fragmentsinclude: a polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 8 from about residue 112 to about residue 213; a polypeptidecomprising an amino acid sequence from about residue 104 to aboutresidue 227 of SEQ ID NO: 8; and a polypeptide comprising an amino acidsequence from about residue 112 to about residue 227 of SEQ ID NO: 8.Other exemplary fragments include polypeptides comprising amino acidsequences of SEQ ID NO: 8 that span, approximately, the followingresidues: 31-213, 31-227, 32-227, 103-217, 103-225, 104-213, 113-213,103-227, 113-227, 131-211, 161-211, 103-225, 227-419, 228-419, 31-419,and 1-419, as described in greater detail below.

The present invention also provides one or more polypeptide precursorsof an Flt4 ligand, wherein one such precursor (designated“prepro-VEGF-C”) comprises the complete amino acid sequence (amino acidresidues 1 to 419) shown in SEQ ID NO: 8. Thus, the invention includes apurified and isolated polypeptide having the amino acid sequence ofresidues 1 to 419 shown in SEQ ID NO: 8. Ligand precursors according tothe invention, when expressed in an appropriate host cell, produce, viacleavage, a polypeptide which binds with high affinity to the Flt4receptor tyrosine kinase. A putative 102 amino acid leader (prepro)peptide has been identified in the amino acid sequence shown in SEQ IDNO: 8. Thus, in a related aspect, the invention includes a purified andisolated polypeptide having the amino acid sequence of residues 103-419shown in SEQ ID NO: 8.

In one embodiment, an expressed Flt4 ligand polypeptide precursor isproteolytically cleaved upon expression to produce an approximately 23kD Flt4 ligand polypeptide. Thus, an Flt4 ligand polypeptide is providedwhich is the cleavage product of the precursor polypeptide shown in SEQID NO: 8 and which has a molecular weight of approximately 23 kD underreducing conditions.

Putative VEGF-C precursors/processing products consisting ofpolypeptides with molecular weights of about 29 and 32 kD also areconsidered aspects of the invention.

In another embodiment, an expressed Flt4 ligand polypeptide precursor isproteolytically cleaved upon expression to produce an approximately 21kD VEGF-C polypeptide. Sequence analysis has indicated that an observed21 kD form has an amino terminus approximately 9 amino acids downstreamfrom the amino terminus of the 23 kD form, suggesting that alternativecleavage sites exist.

From the foregoing, it will be apparent that an aspect of the inventionincludes a fragment of the purified and isolated polypeptide having theamino acid sequence of residues 1 to 419 shown in SEQ ID NO: 8, thefragment being capable of binding with high affinity to Flt4 receptortyrosine kinase. Preferred embodiments include fragments having anapparent molecular weight of approximately 21/23 kD and 29/32 kD asassessed by SDS-PAGE under reducing conditions. More generally, theinvention includes a purified and isolated polypeptide that is a VEGF-Cof vertebrate origin, wherein the VEGF-C has a molecular weight of about21-23 kD, as assessed by SDS-PAGE under reducing conditions, and whereinthe VEGF-C is capable of binding to Flt4 receptor tyrosine kinase(VEGFR-3). Vertebrate VEGF-C forms of about 30-32 kD that are capable ofbinding VEGFR-3 also are intended as an aspect of the invention.

Evidence suggests that the amino acids essential for retaining Flt4ligand activity are contained within approximately amino acids103/112-226/227 of SEQ ID NO: 8, and that a carboxy-terminal proteolyticcleavage to produce a mature, naturally-occurring Flt4 ligand occurs atthe approximate position of amino acids 226-227 of SEQ ID NO: 8.Accordingly, a preferred Flt4 ligand comprises approximately amino acids103-227 of SEQ ID NO: 8.

VEGF-C mutational analysis described herein indicates that a naturallyoccurring VEGF-C polypeptide spanning amino acids 103-227 of SEQ ID NO:8, produced by a natural processing cleavage that defines theC-terminus, exists and is biologically active as an Flt4 ligand. Apolypeptide fragment consisting of residues 104-213 of SEQ ID NO: 8 hasbeen shown to retain VEGF-C biological activity. Additional mutationalanalyses indicate that a polypeptide spanning only amino acids 113-213of SEQ ID NO: 8 retains Flt4 ligand activity. Accordingly, preferredpolypeptides comprise sequences spanning, approximately, amino acidresidues 103-227, 104-213, or 113-213, of SEQ ID NO: 8.

Moreover, sequence comparisons of members of the VEGF family ofpolypeptides provide an indication that still smaller fragments willretain biological activity, and such smaller fragments are intended asaspects of the invention. In particular, eight highly conserved cysteineresidues of the VEGF family of polypeptides define a region from residue131 to residue 211 of SEQ ID NO: 8 (see FIGS. 2, 5 & 10); therefore, apolypeptide spanning from about residue 131 to about residue 211 isexpected to retain VEGF-C biological activity. In fact, a polypeptidecomprising approximately residues 161-211, which retains anevolutionarily-conserved RCXXCC motif, is postulated to retain VEGF-Cactivity, and therefore is intended as an aspect of the invention.

In addition to binding Flt4, VEGF-C polypeptides are shown herein tobind and activate KDR/flk-1 receptor tyrosine kinase (VEGFR-2). Thus,the invention includes a purified and isolated polypeptide that iscapable of binding to at least one of KDR receptor tyrosine kinase(VEGFR-2) and Flt4 receptor tyrosine kinase (VEGFR-3), the polypeptidecomprising a portion of the amino acid sequence in SEQ ID NO: 8effective to permit such binding. In one preferred embodiment, theportion of the amino acid sequence in SEQ ID NO: 8 is a continuousportion having as its amino terminal residue an amino acid betweenresidues 102 and 161 of SEQ ID NO: 8 and having as its carboxy terminalresidue an amino acid between residues 210 and 228 of SEQ ID NO: 8. In ahighly preferred embodiment, the portion has, as its amino terminalresidue, an amino acid between residues 102 and 131 of SEQ ID NO: 8. Ina very highly preferred embodiment, the portion of the amino acidsequence in SEQ ID NO: 8 is a continuous portion having as its aminoterminal residue an amino acid between residues 102 and 114 of SEQ IDNO: 8 and having as its carboxy terminal residue an amino acid betweenresidues 212 and 228 of SEQ ID NO: 8. Polypeptides of the inventionwhich bind to and activate a receptor (e.g., VEGFR-2 or VEGFR-3) areuseful for stimulating VEGF-C biological activities that are mediatedthrough the receptor. Polypeptides of the invention which bind to but donot activate a receptor are useful for inhibiting VEGF-C activitiesmediated through that receptor.

The definition of polypeptides of the invention is intended to includewithin its scope variants thereof. The polypeptide variants contemplatedinclude purified and isolated polypeptides having amino acid sequencesthat differ from the exact amino acid sequences of such polypeptides(e.g., VEGF-C, VEGF-C precursors and VEGF-C fragments) by conservativesubstitutions, as recognized by those of skill in the art, that arecompatible with the retention of at least one VEGF-C biological activityor VEGF-C-inhibitory activity of the polypeptide. The term “variants,”when used to refer to polypeptides, also is intended to includepolypeptides having amino acid additions, including but not limited toadditions of a methionine and/or leader sequence to promote translationand/or secretion; additions of peptide sequences to facilitatepurification (e.g., polyhistidine sequences and/or epitopes for antibodypurification); and additions of polypeptide-encoding sequences toproduce fusion proteins with VEGF-C. The term “variants” also isintended to include polypeptides having amino acid deletions at theamino terminus, the carboxy terminus, or internally of amino acids thatare non-conserved amongst the human, mouse, and quail VEGF-C sequencestaught herein, and that are compatible with the retention of the VEGF-Cor VEGF-C-inhibitory activity of the polypeptide to which the deletionshave been made.

The term “variant” also is intended to include polypeptides havingmodifications to one or more amino acid residues that are compatiblewith retaining VEGF-C or VEGF-C inhibitory activity of the polypeptide.Such modifications include glycosylations (identical or different toglycosylations of native VEGF-C); and the addition of other substituents(e.g., labels, compounds to increase serum half-life (e.g., polyethyleneglycol), and the like.

Additional polypeptides of the invention include certain fragments thathave been observed to result from the processing of prepro-VEGF-C intomature VEGF-C. For example, the invention includes a purified andisolated polypeptide having a molecular weight of about 29 kD asassessed by SDS-PAGE under reducing conditions and having an amino acidsequence consisting essentially of a portion of SEQ ID NO: 8 havingresidue 228 of SEQ ID NO: 8 as its amino terminal amino acid residue;and a purified and isolated polypeptide having a molecular weight ofabout 15 kD as assessed by SDS-PAGE under reducing conditions and havingan amino acid sequence consisting essentially of a portion of SEQ ID NO:8 having residue 32 of SEQ ID NO: 8 as its amino terminal amino acidresidue. Such polypeptides are expected to modulate VEGF-C biologicalactivity through their interactions with VEGF-C receptors and/orinteractions with biologically active VEGF-C.

Some of the conserved cysteine residues in VEGF-C participate ininterchain disulfide bonding to make homo- and heterodimers of thevarious naturally occurring VEGF-C polypeptides. Beyond the precedingconsiderations, evidence exists that VEGF-C polypeptides lackinginterchain disulfide bonds retain VEGF-C biological activity.Consequently, the materials and methods of the invention include allVEGF-C fragments that retain at least one biological activity of VEGF-C,regardless of the presence or absence of interchain disulfide bonds. Theinvention also includes multimers (including dimers) comprising suchfragments linked to each other or to other polypeptides. Fragmentlinkage may be by way of covalent bonding (e.g., disulfide bonding) ornon-covalent bonding of polypeptide chains (e.g, hydrogen bonding,bonding due to stable or induced dipole-dipole interactions, bonding dueto hydrophobic or hydrophilic interactions, combinations of thesebonding mechanisms, and the like). Thus, the invention includes apurified and isolated polypeptide multimer, wherein at least one monomerthereof is a polypeptide that is capable of binding to VEGFR-2 and/orVEGFR-3, the polypeptide comprising a portion of the amino acid sequencein SEQ ID NO: 8 effective to permit such binding, and wherein themultimer itself is capable of binding to VEGFR-2 and/or VEGFR-3. In apreferred embodiment, the multimer has at least one VEGF-C biologicalactivity as taught herein.

In one embodiment, at least one monomer of the multimer is a polypeptidefrom another member of the PDGFNEGF family of proteins, e.g., a vascularendothelial growth factor (VEGF) polypeptide, a vascular endothelialgrowth factor B (VEGF-B) polypeptide, a platelet derived growth factor A(PDGF-A) polypeptide, a platelet derived growth factor B (PDGF-B)polypeptide, a c-fos induced growth factor (FIGF) polypeptide, or aplacenta growth factor (PlGF) polypeptide.

In a highly preferred embodiment, the multimer of the invention is adimer of two monomer polypeptides. For example, the invention includes adimer wherein each monomer thereof is capable of binding to at least oneof VEGFR-2 and VEGFR-3 and has an amino acid sequence comprising aportion of SEQ ID NO: 8 effective to permit such binding. Dimers havingcovalent attachments and dimers wherein the two monomers are free ofcovalent attachments to each other are contemplated.

In yet another aspect, the invention includes analogs of thepolypeptides of the invention. The term “analog” refers to polypeptideshaving alterations involving one or more amino acid insertions, internalamino acid deletions, and/or non-conservative amino acid substitutions(replacements). The definition of analog is intended to include withinits scope variants of analog polypeptides embodying such alterations.The term “mutant,” when used with respect to polypeptides herein, isintended to refer generically to VEGF-C variants, VEGF-C analogs, andvariants of VEGF-C analogs. Preferred analogs possess at least 90% aminoacid sequence similarity to the native peptide sequence from which theanalogs were derived. Highly preferred analogs possess 95%, 96%, 97%,98%, 99%, or greater amino acid sequence similarity to the nativepeptide sequence.

For example, in one embodiment, the invention includes a polypeptideanalog of a VEGF-C of vertebrate origin that is capable of binding toVEGFR-3 (e.g., an analog of a vertebrate VEGF-C of about 21-23 kD asassessed by SDS-PAGE under reducing conditions), wherein anevolutionarily conserved cysteine residue in the VEGF-C has been deletedor replaced, and wherein the analog is capable of binding to VEGFR-3 andhas reduced VEGFR-2 binding affinity relative to the wildtype VEGF-C.For analogs according to this embodiment of the invention, thedetermination that a residue is “evolutionarily conserved” is madesolely by reference to the alignment of human, mouse, and quail VEGF-Csequences provided herein and aligned to show similarity in FIG. 5. Thepresence of the same residue in all three sequences indicates that theresidue is evolutionarily conserved, notwithstanding the fact thatVEGF-C from other species may lack the residue. In a preferredembodiment, the conserved cysteine residue corresponds to the cysteineat position 156 of SEQ ID NO: 8. “Correspondence to the cysteine atposition 156” is readily determined from an analysis of the vertebrateVEGF-C sequence of interest, since the cysteine at position 156 of SEQID NO: 8 (human VEGF-C) falls within an evolutionarily conserved portionof VEGF-C (see FIG. 5, comparing human, mouse, and quail VEGF-Cpolypeptides). Alignment of human VEGF-C allelic variants, othermammalian VEGF-C polypeptides, and the like with the three VEGF-C formsin FIG. 5 will identify that cysteine which corresponds to the cysteineat position 156 of SEQ ID NO: 8, even if the allelic variant has greateror fewer than exactly 155 residues preceding the cysteine of interest.

In another embodiment, the invention includes a purified polypeptidethat is an analog of human VEGF-C and that is capable of binding to atleast one of Flt-1 receptor tyrosine kinase (VEGFR-1), KDR receptortyrosine kinase (VEGFR-2), and Flt4 receptor tyrosine kinase (VEGFR-3).

Specifically contemplated is an analog of human VEGF-C that bindsVEGFR-3 but has reduced VEGFR-2 binding affinity, as compared to theVEGFR-2 binding affinity of a wildtype human VEGF-C (e.g., as comparedto the VEGFR-2 binding affinity of a human VEGF-C having an amino acidsequence consisting essentially of amino acids 103-227 of SEQ ID NO: 8).One such family of human VEGF-C analogs are VEGF-C Δ₁₅₆ polypeptides. By“VEGF-C ΔC₁₅₆ polypeptide” is meant an analog wherein the cysteine atposition 156 of SEQ ID NO: 8 has been deleted or replaced by anotheramino acid. A VEGF-C ΔC₁₅₆ polypeptide analog can be made from anyVEGF-C polypeptide of the invention that comprises all of SEQ ID NO: 8or a portion thereof that includes position 156 of SEQ ID NO: 8.Preferably, the VEGF-C ΔC₁₅₆ polypeptide analog comprises a portion ofSEQ ID NO: 8 effective to permit binding to VEGFR-3.

For example, the invention includes a VEGF-C ΔC₁₅₆ polypeptide thatbinds VEGFR-3, has reduced VEGFR-2 binding affinity, and has an aminoacid sequence which includes amino acids 131 to 211 of SEQ ID NO: 8,wherein the cysteine residue at position 156 of SEQ ID NO: 8 has beendeleted or replaced. In a preferred embodiment, the VEGF-C ΔC₁₅₆polypeptide comprises a continuous portion of SEQ ID NO: 8, the portionhaving as its amino terminal residue an amino acid between residues 102and 114 of SEQ ID NO: 8, and having as its carboxy terminal residue anamino acid between residues 212 and 228 of SEQ ID NO: 8, wherein thecysteine residue at position 156 of SEQ ID NO: 8 has been deleted orreplaced. In an embodiment exemplified herein, the cysteine residue atposition 156 of SEQ ID NO: 8 has been replaced by a serine residue.

A second family of human VEGF-C analogs that bind VEGFR-3 but havereduced VEGFR-2 binding affinity are VEGF-C ΔR₂₂₆ΔR₂₂₇ polypeptides. By“VEGF-C ΔR₂₂₆ΔR₂₂₇ polypeptide” is meant an analog wherein the arginineresidues at positions 226 and 227 of SEQ ID NO: 8 have been deleted orreplaced by other amino acids, for the purpose of eliminating aproteolytic processing site of the carboxy terminal pro-peptide ofVEGF-C. Preferably, the VEGF-C ΔR₂₂₆ΔR₂₂₇ polypeptide comprises aportion of SEQ ID NO: 8 effective to permit binding of VEGFR-3. Forexample, the invention includes a VEGF-C ΔR₂₂₆ΔR₂₂₇ polypeptide havingan amino acid sequence comprising amino acids 112-419 of SEQ ID NO: 8,wherein the arginine residues at positions 226 and 227 of SEQ ID NO: 8have been deleted or replaced. Specifically exemplified herein is aVEGF-C ΔR₂₂₆ΔR227 polypeptide wherein the arginine residues at positions226 and 227 of SEQ ID NO: 8 have been replaced by serine residues.

Another family of VEGF-C analogs of the invention are humanVEGF-C^(basic) polypeptides. By “VEGF-C^(basic) polypeptide” is meant aVEGF-C analog wherein at least one amino acid having a basic side chainhas been introduced into the VEGF-C coding sequence, to emulate one ormore basic residues in VEGF (e.g., residues Arg₁₀₈, Lys₁₁₀, and His₁₁₂in the VEGF165 precursor shown in FIG. 2) that have been implicated inVEGF receptor binding. Preferably, two or three basic residues areintroduced into VEGF-C. Based on the VEGF/VEGF-C polypeptide alignmentprovided herein, positions 187, 189, and 191 of SEQ ID NO: 8 arepreferred positions to introduce basic residues. For example, theinvention includes a VEGF-C^(basic) polypeptide that is capable ofbinding to at least one of VEGFR-1, VEGFR-2, and VEGFR-3, and that hasan amino acid sequence comprising residues 131 to 211 of SEQ ID NO: 8,wherein the glutamic acid residue at position 187, the threonine residueat position 189, and the proline residue at position 191 of SEQ ID NO: 8have been replaced by an arginine residue, a lysine residue, and ahistidine residue, respectively.

In yet another aspect of the invention, VEGF-C structural information isemployed to create useful analogs of VEGF. For example, mature VEGF-Ccontains an unpaired cysteine (position 137 of SEQ ID NO: 8) and is ableto form non-covalently bonded polypeptide dimers. In one embodiment, aVEGF analog is created wherein this unpaired cysteine residue frommature VEGF-C is introduced at an analogous position of VEGF (e.g.,introduced in place of Leu₅₈ of the human VEGF165 precursor (FIG. 2,Genbank Acc. No. M32977). Such VEGF analogs are termed VEGF^(+cys)polypeptides. Thus, the invention includes a human VEGF analog wherein acysteine residue is introduced in the VEGF amino acid sequence at aposition selected from residues 53 to 63 of the human VEGF165 precursorhaving the amino acid sequence set forth in SEQ ID NO: 56. At least fournaturally occurring VEGF isoforms have been described, and VEGF^(+cys)polypeptide analogs of each isoform are contemplated. Most preferably,the cysteine is introduced at a position in a VEGF isoform whichcorresponds to position 58 of the VEGF165 precursor having the aminoacid sequence set forth in SEQ ID NO: 56.

The present invention also provides purified and isolatedpolynucleotides (i.e., nucleic acids) encoding all of the polypeptidesof the invention, including but not limited to cDNAs and genomic DNAsencoding VEGF-C precursors, VEGF-C, and biologically active fragmentsthereof, and DNAs encoding VEGF-C variants and VEGF-C analogs. Apreferred nucleic acid of the invention comprises a DNA encoding aminoacid residues 1 to 419 of SEQ ID NO: 8 or one of the aforementionedfragments or analogs thereof. Due to the degeneracy of the genetic code,numerous such coding sequences are possible, each having in common thecoding of the amino acid sequence shown in SEQ ID NO: 8 or the fragmentor analog thereof. Distinct polynucleotides encoding any polypeptide ofthe invention by virtue of the degeneracy of the genetic code are withinthe scope of the invention.

A preferred polynucleotide according to the invention comprises thehuman VEGF-C cDNA sequence set forth in SEQ ID NO: 7 from nucleotide 352to 1611. Other polynucleotides according to the invention encode aVEGF-C polypeptide from, e.g., mammals other than humans, birds (e.g.,avian quails), and others. Still other polynucleotides of the inventioncomprise a coding sequence for a VEGF-C fragment, and allelic variantsof those DNAs encoding part or all of VEGF-C.

Still other polynucleotides of the invention comprise a coding sequencefor a VEGF-C variant or a VEGF-C analog. Preferred variant-encoding andanalog-encoding polynucleotides comprise the human, mouse, or quailVEGF-C cDNA sequences disclosed herein (e.g., nucleotides 352-1611 ofSEQ ID NO: 7 or continuous portions thereof) wherein one or more codonsubstitutions, deletions, or insertions have been introduced to createthe variant/analog-encoding polynucleotide. For example, a preferredpolynucleotide encoding a VEGF-C ΔC₁₅₆ polypeptide comprises all or aportion of SEQ ID NO: 7 wherein the cysteine codon at positions 817-819has been replaced by a codon encoding a different amino acid (e.g., aserine-encoding TCC codon).

The invention further comprises polynucleotides that hybridize to theaforementioned polynucleotides under standard stringent hybridizationconditions. Exemplary stringent hybridization conditions are as follows:hybridization at 42° C. in 50% formamide, 5×SSC, 20 mM Na.PO₄, pH 6.8;and washing in 0.2×SSC at 55° C. It is understood by those of skill inthe art that variation in these conditions occurs based on the lengthand GC nucleotide content of the sequences to be hybridized. Formulasstandard in the art are appropriate for determining appropriatehybridization conditions. See Sambrook et al., Molecular Cloning: ALaboratory Manual (Second ed., Cold Spring Harbor Laboratory Press,1989) §§9.47-9.51. These polynucleotides, capable of hybridizing topolynucleotides encoding VEGF-C, VEGF-C fragments, or VEGF-C analogs,are useful as nucleic acid probes for identifying, purifying andisolating polynucleotides encoding other (non-human) mammalian forms ofVEGF-C and human VEGF-C allelic variants. Additionally, thesepolynucleotides are useful in screening methods of the invention, asdescribed below.

Preferred nucleic acids useful as probes of the invention comprisenucleic acid sequences of at least about 16 continuous nucleotides ofSEQ ID NO: 7. More preferably, these nucleic acid probes would have atleast about 20 continuous nucleotides found in SEQ ID NO: 7. In usingthese nucleic acids as probes, it is preferred that the nucleic acidsspecifically hybridize to a portion of the sequence set forth in SEQ IDNO: 7. Specific hybridization is herein defined as hybridization understandard stringent hybridization conditions. To identify and isolateother mammalian VEGF-C genes specifically, nucleic acid probespreferably are selected such that they fail to hybridize to genesrelated to VEGF-C (e.g., fail to hybridize to human VEGF or to humanVEGF-B genes).

Thus, the invention comprehends polynucleotides comprising at leastabout 16 nucleotides wherein the polynucleotides are capable ofspecifically hybridizing to a gene encoding VEGF-C, e.g., a human gene.The specificity of hybridization ensures that a polynucleotide of theinvention is able to hybridize to a nucleic acid encoding a VEGF-C underhybridization conditions that do not support hybridization of thepolynucleotide to nucleic acids encoding, e.g., VEGF or VEGF-B. In oneembodiment, polynucleotides of at least about 16 nucleotides, andpreferably at least about 20 nucleotides, are selected as continuousnucleotide sequences found in SEQ ID NO: 7 or the complement of thenucleotide sequence set forth in SEQ ID NO: 7.

In another embodiment, the invention includes polynucleotides having atleast 90 percent (preferably at least 95 percent, and more preferably atleast 97, 98, or 99 percent) nucleotide sequence identity with anucleotide sequence encoding a polypeptide of the invention. In a highlypreferred embodiment, the polynucleotides have at least 95 percentsequence identity with a nucleotide sequence encoding a human VEGF-Cprecursor (such as the VEGF-C precursor in SEQ ID NO: 8 and allelicvariants thereof), human VEGF-C, or biologically active VEGF-Cfragments.

Additional aspects of the invention include vectors which comprisenucleic acids of the invention; and host cells transformed ortransfected with nucleic acids or vectors of the invention. Preferredvectors of the invention are expression vectors wherein nucleic acids ofthe invention are operatively connected to appropriate promoters andother control sequences that regulate transcription and/or subsequenttranslation, such that appropriate prokaryotic or eukaryotic host cellstransformed or transfected with the vectors are capable of expressingthe polypeptide encoded thereby (e.g., the VEGF-C, VEGF-C fragment,VEGF-C variant, or VEGF-C analog encoded thereby). A preferred vector ofthe invention is plasmid pFLT4-L, having ATCC accession no. 97231. Suchvectors and host cells are useful for recombinantly producingpolypeptides of the invention, including VEGF-C, and fragments,variants, and analogs thereof.

In a related aspect of the invention, host cells such as procaryotic andeukaryotic cells, especially unicellular host cells, are modified toexpress polypeptides of the invention. Host cells may be stablytransformed or transfected with isolated DNAs of the invention in amanner allowing expression of polypeptides of the invention therein.Thus, the invention further includes a method of making polypeptides ofthe invention. In a preferred method, a nucleic acid or vector of theinvention is expressed in a host cell, and a polypeptide of theinvention is purified from the host cell or the host cell's growthmedium.

Similarly, the invention includes a method of making a polypeptidecapable of specifically binding to VEGFR-1, VEGFR-2 and/or VEGFR-3,comprising the steps of: (a) transforming or transfecting a host cellwith a nucleic acid of the invention; (b) cultivating the host cell toexpress the nucleic acid; and (c) purifying a polypeptide capable ofspecifically binding to VEGFR-1, VEGFR-2, and/or VEGFR-3 from the hostcell or from the host cell's growth media. The invention also includespurified and isolated polypeptides produced by methods of the invention.In one preferred embodiment, the invention includes a human VEGF-Cpolypeptide or biologically active fragment, variant, or analog thereofthat is substantially free of other human polypeptides.

Alternatively, host cells may be modified by activating an endogenousVEGF-C gene that is not normally expressed in the host cells or that isexpressed at a lower rate than is desired. Such host cells are modified(e.g., by homologous recombination) to express the VEGF-C by replacing,in whole or in part, the naturally-occurring VEGF-C promoter with partor all of a heterologous promoter so that the host cells express VEGF-C.In such host cells, the heterologous promoter DNA is operatively linkedto the VEGF-C coding sequences, i.e., controls transcription of theVEGF-C coding sequences. See, for example, PCT International PublicationNo. WO 94/12650; PCT International Publication No. WO 92/20808; and PCTInternational Publication No. WO 91/09955. The invention alsocontemplates that, in addition to heterologous promoter DNA, amplifiablemarker DNA (e.g., ada, dhfr, and the multifunctional CAD gene whichencodes carbamyl phosphate synthase, aspartate transcarbamylase, anddihydro-orotase) and/or intron DNA may be recombined along with theheterologous promoter DNA into the host cells. If linked to the VEGF-Ccoding sequences, amplification of the marker DNA by standard selectionmethods results in co-amplification of the VEGF-C coding sequences insuch host cells. Thus, the invention includes, for example, a cellcomprising a nucleic acid having a sequence encoding human VEGF-C andfurther comprising a non-VEGF-C promoter sequence (i.e., a heterologouspromoter sequence) or other non-VEGF-C control sequence that increasesRNA transcription in the cell of the sequence encoding human VEGF-C.

The DNA sequence information provided by the present invention alsomakes possible the development, by homologous recombination or“knockout” strategies [see, Capecchi, Science, 244: 1288-1292 (1989)],of rodents that fail to express functional VEGF-C or that express aVEGF-C fragment, variant, or analog. Such rodents are useful as modelsfor studying the activities of VEGF-C and VEGF-C modulators in vivo.

In another aspect, the invention includes an antibody that specificallybinds to one or more polypeptides of the invention, and/or binds topolypeptide multimers of the invention. In the context of antibodies ofthe invention, the term “specifically binds” is intended to excludeantibodies that cross-react with now-identified, related growth factors,such as VEGF, VEGF-B, PDGF-A, PDGF-B, FIGF, and PlGF. However, due tothe high level of amino acid similarity shared by VEGF-C polypeptides ofdifferent species, it will be understood that antibodies thatspecifically bind to human VEGF-C polypeptides of the invention will, inmany instances, also bind non-human (e.g., mouse, quail) VEGF-Cpolypeptides of the invention. Antibodies, both monoclonal andpolyclonal, may be made against a polypeptide of the invention accordingto standard techniques in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988)). Standard protein manipulationtechniques and recombinant techniques also may be employed to generatehumanized antibodies and antigen-binding antibody fragments and otherchimeric antibody polypeptides, all of which are considered antibodiesof the invention. The invention further includes hybridoma cells thatproduce antibodies of the invention or other cell types that have beengenetically engineered to express antibody polypeptides of theinvention. Antibodies of the invention may be used in diagnosticapplications to to monitor angiogenesis, vascularization, lymphaticvessels and their disease states, wound healing, or certain tumor cells,hematopoietic, or leukemia cells. The antibodies also may be used toblock the ligand from activating its receptors; to purify polypeptidesof the invention; and to assay fluids for the presence of polypeptidesof the invention. The invention further includes, immunological assays(including radio-immuno assays, enzyme linked immunosorbent assays,sandwich assays and the like) which employ antibodies of the invention.

Ligands according to the invention may be labeled with a detectablelabel and used to identify their corresponding receptors in situ.Labeled Flt4 ligand and anti-Flt4 ligand antibodies may be used asimaging agents in the detection of lymphatic vessels, high endothelialvenules and their disease states, and Flt4 receptors expressed inhistochemical tissue sections. The ligand or antibody may be covalentlyor non-covalently coupled to a suitable supermagnetic, paramagnetic,electron dense, echogenic, or radioactive agent for imaging. Other,non-radioactive labels, such as biotin and avidin, may also be used.

A related aspect of the invention is a method for the detection ofspecific cells, e.g., endothelial cells. These cells may be found invivo, or in ex vivo biological tissue samples. The method of detectioncomprises the steps of contacting a biological tissue comprising, e.g.,endothelial cells, with a polypeptide according to the invention whichis capable of binding to VEGFR-2 and/or VEGFR-3, under conditionswherein the polypeptide binds to the cells, optionally washing thebiological tissue, and detecting the polypeptide bound to the cells inthe biological tissue, thereby detecting the cells. It will be apparentthat certain polypeptides of the invention are useful for detectingand/or imaging cells that express both VEGFR-2 and VEGFR-3, whereasother polypeptides (e.g., VEGF-C ΔC₁₅₆ polypeptides) are useful forimaging specifically those cells which express VEGFR-3.

The many biological activities described herein for VEGF-C (includingbut not limited to affecting growth and migration of vascularendothelial cells; promoting growth of lymphatic endothelial cells andlymphatic vessels; increasing vascular permeability; and affectingmyelopoiesis (e.g., growth of neutrophilic granulocytes)) 0.10 supportnumerous diagnostic and in vitro and in vivo clinical utilities forpolypeptides and antibodies of the invention, for modulating(stimulating or inhibiting) these biological activities. Generally,VEGF-C and precursor, fragment, variant, and analog polypeptides thatretain one or more VEGF-C biological activities are useful agonists forstimulating the desired biological activity; whereas precursor,fragment, variant, and analog polypeptides that are capable of bindingto VEGFR-2 and/or VEGFR-3 (either alone or as a homo- or hetero-dimerwith other polypeptides) without stimulating receptor-mediated VEGF-Cactivity (i.e., without activating the receptor) are useful asantagonists (inhibitors) of VEGF-C. Similarly, antibodies of theinvention that bind biologically active VEGF-C forms and therebyinterfere with VEGF-C-receptor interactions are useful as inhibitors ofVEGF-C. Antisense oligonucleotides comprising a portion of the VEGF-Ccoding sequence and/or its complement also are contemplated asinhibitors of the invention. Both biologically active polypeptides andinhibitor polypeptides of the invention have utilities in variousimaging applications.

For example, the biological effects of VEGF-C on vascular endothelialcells indicate in vivo uses for polypeptides of the invention forstimulating angiogenesis (e.g., during wound healing, in tissuetransplantation, in eye diseases, in the formation of collateral vesselsaround arterial stenoses and into injured tissues after infarction) andfor inhibiting angiogenesis (e.g., to inhibit tumor growth and/ormetastatic cancer). The biological effects on vascular endothelial cellsindicate in vitro uses for biologically active forms of VEGF-C topromote the growth of (including proliferation of) cultured vascularendothelial cells and precursors thereof.

The biological effects of VEGF-C on lymphatic endothelia indicate invivo uses for polypeptides of the invention for stimulatinglymphangiogenesis (e.g., to promote re-growth or permeability oflymphatic vessels in, for example, organ transplant patients; tomitigate the loss of axillary lymphatic vessels following surgicalinterventions in the treatment of cancer (e.g., breast cancer); to treataplasia of the lymphatic vessels or lymphatic obstructions) and forinhibiting it (e.g., to treat lymphangiomas). Additional in vivo usesfor polypeptides of the invention include the treatment or prevention ofinflammation, edema, elephantiasis, and Milroy's disease. The biologicaleffects on lymphatic endothelial cells indicate in vitro uses forbiologically active forms of VEGF-C to promote the growth of culturedlymphatic endothelial cells and precursors thereof.

Thus, the invention includes a method of modulating(stimulating/increasing or inhibiting/decreasing) the growth ofvertebrate endothelial cells or vertebrate endothelial precursor cellscomprising contacting such endothelial cells or precursor cells with apolypeptide or antibody (or antigen-binding portion thereof) of theinvention, in an amount effective to modulate the growth of theendothelial or endothelial precursor cells. Mammalian endothelial cellsand their precursors are preferred. Human endothelial cells are highlypreferred. In one embodiment, the endothelial cells are lymphaticendothelial cells. In another embodiment, the cells are vascularendothelial cells. The method may be an in vitro method (e.g., forcultured endothelial cells) or an in vivo method. The in vitro growthmodulation of CD34+ endothelial precursor cells [see, e.g., Asahara etal., Science, 275:964-967 (1997)] isolated from peripheral blood, bonemarrow, or cord blood is specifically contemplated. For in vivo methods,it is highly preferable to administer a pharmaceutical composition(comprising the polypeptide formulated in a pharmaceutically acceptablediluent, adjuvant, excipient, carrier, or the like) to the subject, inan amount effective to modulate the growth of lymphatic endothelialcells in vivo.

In one preferred embodiment, the endothelial cells are lymphaticendothelial cells, and the polypeptide is one that has reduced effect onthe permeability of mammalian blood vessels compared to a wildtypeVEGF-C polypeptide (e.g., compared with VEGF-C having an amino acidsequence set forth in SEQ ID NO: 8 from residue 103 to residue 227).VEGF-C ΔC₁₅₆ polypeptides are contemplated for use in this embodiment.

In modulating the growth of endothelial cells in vivo, the inventioncontemplates the modulation of endothelial cell-related disorders.Endothelial cell disorders contemplated by the invention include, butare not limited to, physical loss of lymphatic vessels (e.g., surgicalremoval of axillary lymph tissue), lymphatic vessel occlusion (e.g.,elephantiasis), and lymphangiomas. In a preferred embodiment, thesubject, and endothelial cells, are human. The endothelial cells may beprovided in vitro or in vivo, and they may be contained in a tissuegraft. An effective amount of a polypeptide is defined herein as thatamount of polypeptide empirically determined to be necessary to achievea reproducible change in cell growth rate (as determined by microscopicor macroscopic visualization and estimation of cell doubling time, ornucleic acid synthesis assays), as would be understood by one ofordinary skill in the art.

Polypeptides of the invention may be used to stimulate lymphocyteproduction and maturation, and to promote or inhibit trafficking ofleukocytes between tissues and lymphatic vessels or to affect migrationin and out of the thymus.

The biological effects of VEGF-C on myelopoiesis indicate in vivo and invitro uses for polypeptides of the invention for stimulatingmyelopoiesis (especially growth of neutrophilic granuloctyes) orinhibiting it. Thus, the invention includes a method for modulatingmyelopoiesis in a mammalian subject comprising administering to amammalian subject in need of modulation of myelopoiesis an amount of apolypeptide or antibody (or antigen-binding portion thereof) of theinvention that is effective to modulate myelopoiesis. In one embodiment,a mammalian subject suffering from granulocytopenia is selected, and themethod comprises administering to the subject an amount of a polypeptideeffective to stimulate myelopoiesis. In particular, a polypeptide of theinvention is administered in an amount effective to increase theneutrophil count in blood of the subject. Preferred subjects are humansubjects. An effective amount of a polypeptide is an amount ofpolypeptide empirically determined to be necessary to achieve areproducible change in the production of neutrophilic granulocytes (asdetermined by microscopic or macroscopic visualization and estimation ofcell doubling time, or nucleic acid synthesis assays), as would beunderstood by one of ordinary skill in the art.

In a related embodiment, the invention includes a method of increasingthe number of neutrophils in the blood of a mammalian subject comprisingthe step of expressing in a cell in a subject in need of an increasednumber of blood neutrophils a DNA encoding a VEGF-C protein, the DNAoperatively linked to a non-VEGF-C promoter or other non-VEGF-C controlsequence that promotes expression of the DNA in the cell.

Similarly, the invention includes a method of modulating the growth ofneutrophilic granulocytes in vitro or in vivo comprising the step ofcontacting mammalian stem cells with a polypeptide or antibody of theinvention in an amount effective to modulate the growth of mammalianendothelial cells.

More generally, the invention includes a method for modulating thegrowth of CD34+ progenitor cells (especially hematopoietic progenitorcells and endothelial progenitor cells) in vitro or in vivo comprisingthe step of contacting mammalian CD34+ progenitor cells with apolypeptide or antibody of the invention in an amount effective tomodulate the growth of mammalian endothelial cells. For in vitromethods, CD34+ progenitor cells isolated from cord blood or bone marroware specifically contemplated.

It will be apparent from the Detailed Description below that in vitroand in vivo methods of the invention for stimulating the growth of CD34+precursor cells also include methods wherein polypeptides of theinvention are employed together (simultaneously or sequentially) withother polypeptide factors for the purpose of modulatinghematopoiesis/myelopoiesis or endothelial cell proliferation. Such otherfactors include, but are not limited to colony stimulating factors(“CSFs,” e.g., granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), andgranulocyte-macrophage-CSF (GM-CSF)), interleukin-3 (IL-3, also calledmulti-colony stimulating factor), other interleukins, stem cell factor(SCF), other polypeptide factors, such as VEGF, and their analogs thathave been described and are known in the art. See generally The CytokineHandbook, Second Ed., Angus Thomson (editor), Academic Press (1996);Callard and Gearing, The Cytokine FactsBook, Academic Press Inc. (1994);and Cowling and Dexter, TIBTECH, 10(10):349-357 (1992). The use of apolypeptide of the invention as a progenitor cell or myelopoietic cellgrowth factor or co-factor with one or more of the foregoing factors maypotentiate previously unattainable myelopoietic effects and/orpotentiate previously attainable myelopoietic effects while using lessof the foregoing factors than would be necessary in the absence of apolypeptide of the invention.

In addition to methods, the invention includes compositions comprisingpolypeptides of the invention in admixture with one or more of thefactors identified in the previous paragraph. Preferred compositionsfurther comprise a pharmaceutically acceptable diluent, adjuvant,excipient, or carrier. The invention also includes kits comprising (a)at least one polypeptide of the invention packaged with (b) one or moreof the foregoing polypeptides (e.g., in unit dosage form, but not inadmixture with each other).

For methods which involve the in vivo administration of polypeptides orantibodies of the invention, it is contemplated that the polypeptides orantibodies will be administered in any suitable manner using anappropriate pharmaceutically-acceptable vehicle, e.g., apharmaceutically-acceptable diluent, adjuvant, excipient or carrier.Thus, the invention further includes compositions, e.g., pharmaceuticalcompositions, comprising one or more polypeptides or antibodies of theinvention. By pharmaceutical composition is meant a composition that maybe administered to a mammalian host, e.g., orally, topically,parenterally (including subcutaneous injections, intravenous,intramuscular, intracisternal injection or infusion techniques), byinhalation spray, or rectally, in unit dosage formulations containingconventional non-toxic carriers, diluents (e.g., calcium carbonate,sodium carbonate, lactose, calcium phosphate, sodium phosphate, kaolin,water), adjuvants, vehicles, and the like, including but not limited toflavoring agents, preserving agents; granulating and disintegratingagents; binding agents; time delay materials; oils; suspending agents;dispersing or wetting agents; anti-oxidants; emulsifiers, etc.

The invention further provides a method of using a polypeptide of theinvention for the manufacture of a medicament for use in any of theforegoing methods. Similarly, the invention further provides a method ofusing a polypeptide of the invention for the manufacture of a medicamentfor the treatment of any of the foregoing indicated conditions anddisease states. Such methods optionally involve the use of additionalbiologically active ingredients (e.g., VEGF, PlGF, G-CSF, etc.) for themanufacture of the medicament.

Effective amounts of polypeptides for the foregoing methods areempirically determined using standard in vitro and in vivo dose-responseassays. In addition, experimental data provided herein provide guidanceas to amounts of polypeptides of the invention that are effective forachieving a desired biological response. For example, the dissociationconstants determined for one form of mature VEGF-C (K_(D)=135 pM forVEGFR-3 and K_(D)=410 pM for VEGFR-2) provide an indication as to theconcentration of VEGF-C necessary to achieve biological effects, becausesuch dissociation constants represent concentrations at which half ofthe VEGF-C polypeptide is bound to the receptors through which VEGF-Cbiological effects are mediated. Results from in vivo Miles assays,wherein 0-8 picomoles of VEGF-C was injected intradermally, provide anindication that picomole quantities of mature VEGF-C are sufficient toinduce localized biological effects. In vitro analysis of ³H-thymidineincorporation into bovine capillary endothelial cells treated with amature VEGF-C form showed increasing VEGF-C effects on cellproliferation at concentrations of 10-1000 pM. Collectively, this datasuggests that localized concentrations of 100-1000 pM of fully-processedVEGF-C have VEGF-C biological activity in vivo. Effective concentrationsof other polypeptides of the invention are generally expected tocorrelate with the dissociation constant of the polypeptides for therelevant receptors. Pharmacokinetic and pharmacological analyses revealsthe preferred dosages, dosage formulations, and methods ofadministration to achieve the desired local or systemic concentration ofa polypeptide of the invention.

Polypeptides of the invention also may be used to quantify futuremetastatic risk by assaying biopsy material for the presence of activereceptors or ligands in a binding assay. Such a binding assay mayinvolve the use of a detectably labeled polypeptide of the invention orof an unlabeled polypeptide in conjunction with a labeled antibody, forexample. Kits comprising such substances are included within the scopeof the invention.

The present invention also provides methods for using the claimednucleic acids (i.e., polynucleotides) in screening for endothelial celldisorders. In a preferred embodiment, the invention provides a methodfor screening an endothelial cell disorder in a mammalian subjectcomprising the steps of providing a sample of endothelial cell nucleicacids from the subject, contacting the sample of endothelial cellnucleic acids with a polynucleotide of the invention which is capable ofhybridizing to a gene encoding VEGF-C (and preferably capable ofhybridizing to VEGF-C mRNA), determining the level of hybridizationbetween the endothelial cell nucleic acids and the polynucleotide, andcorrelating the level of hybridization with a disorder. A preferredmammalian subject, and source of endothelial cell nucleic acids, is ahuman. The disorders contemplated by the method of screening withpolynucleotides include, but are not limited to, vessel disorders suchas the aforementioned lymphatic vessel disorders, and hypoxia.

Purified and isolated polynucleotides encoding other (non-human) VEGF-Cforms also are aspects of the invention, as are the polypeptides encodedthereby, and antibodies that bind to non-human VEGF-C forms. Preferrednon-human forms of VEGF-C are forms derived from other vertebratespecies, including avian and mammalian species. Mammalian forms arehighly preferred. Thus, the invention includes a purified and isolatedmammalian VEGF-C polypeptide, and also a purified and isolatedpolynucleotide encoding such a polypeptide.

In one embodiment, the invention includes a purified and isolatedpolypeptide having the amino acid sequence of residues 1 to 415 of SEQID NO: 11, which sequence corresponds to a putative mouse VEGF-Cprecursor. The putative mouse VEGF-C precursor is believed to beprocessed into a mature mouse VEGF-C in a manner analogous to theprocessing of the human prepro-polypeptide. Thus, in a related aspect,the invention includes a purified and isolated polypeptide capable ofbinding with high affinity to an Flt4 receptor tyrosine kinase (e.g., ahuman or mouse Flt-4 receptor tyrosine kinase), the polypeptidecomprising a fragment of the purified and isolated polypeptide havingthe amino acid sequence of residues 1 to 415 of SEQ ID NO: 11, thefragment being capable of binding with high affinity to the Flt4receptor tyrosine kinase. The invention further includes multimers ofthe foregoing polypeptides and purified and isolated nucleic acidsencoding the foregoing polypeptides, such as a nucleic acid comprisingall or a portion of the sequence shown in SEQ ID NO: 10.

In another embodiment, the invention includes a purified and isolatedquail VEGF-C polypeptide, biologically active fragments and multimersthereof, and polynucleotides encoding the foregoing polypeptides.

It is also contemplated that VEGF-C polypeptides from other species maybe altered in the manner described herein with respect to human VEGF-Cvariants, in order to alter biological properties of the wildtypeprotein. For example, elimination of the cysteine at position 152 of SEQID NO: 11 or position 155 of SEQ ID NO: 13 is expected to alter VEGFR-2binding properties in the manner described below for human VEGF-C ΔC₁₅₆mutants.

In yet another embodiment, the invention includes a DNA comprising aVEGF-C promoter, that is capable of promoting expression of a VEGF-Cgene or another operatively-linked, protein-encoding gene in native hostcells, under conditions wherein VEGF-C is expressed in such cells. Thus,the invention includes a purified nucleic acid comprising a VEGF-Cpromoter sequence. Genomic clone lambda 5 described herein comprisesmore than 5 kb of human genomic DNA upstream of the VEGF-C translationinitiation codon, and contains promoter DNA of the invention.Approximately 2.4 kb of this upstream sequence is set forth in SEQ IDNO: 48. Thus, in one embodiment, the invention includes a purifiednucleic acid comprising a portion of SEQ ID NO: 48, wherein the portionis capable of promoting expression of a protein encoding geneoperatively linked thereto under conditions wherein VEGF-C is expressedin native host cells. Similarly, the invention includes a chimericnucleic acid comprising a VEGF-C promoter nucleic acid according to theinvention operatively connected to a sequence encoding a protein otherthan a human VEGF-C.

Additional aspects and embodiments of the invention will be apparentfrom the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 schematically depicts major endothelial cell receptor tyrosinekinases and growth factors involved in vasculogenesis and angiogenesis.Major structural domains are depicted, including immunoglobulin-likedomains (IGH), epidermal growth factor homology domains (EGFH),fibronectin type III domains (FNIII), transmembrane (TM) andjuxtamembrane (JM) domains, tyrosine kinase (TK1, TK2) domains, kinaseinsert domains (KI), and carboxy-terminal domains (CT).

FIG. 2 shows a comparison of the deduced amino acid sequences of PDGF-A(SEQ ID NO: 53), PDGF-B (SEQ ID NO: 54), PlGF-1 (SEQ ID NO: 55),VEGF-B₁₆₇ (SEQ ID NO: 57), VEGF165 (SEQ ID NO: 56), and Flt4 ligand(VEGF-C, (SEQ ID NO: 8)).

FIG. 3 schematically depicts the VEGF-C promoter-reporter constructs andtheir activities in transfected HeLa cells. A restriction map of aportion of a genomic clone that includes the VEGF-C initiation codon andabout 6 kb of upstream sequence is depicted above the constructs.Constructs were made linking putative VEGF-C promoter to the Luciferasereporter gene in pGL3 vector (Promega) and introduced into HeLa cells bycalcium phosphate-mediated transfection method. The Luciferase activityobtained was compared to the level using the promoterless pGL3basicconstruct to obtain a measure of relative promoter activity. Luciferaseactivity is expressed graphically as a ratio of activity of theconstructs versus this control. Also shown are numerical ratios ofLuciferase activity in experiments where the constructs were transfectedinto HeLa cells and cells were starved 24 hours followed by serumstimulation for four hours (Luciferase activity is expressed as a ratioof activity in serum-stimulated versus serum-starved cells).

FIG. 4 graphically depicts the results of a competitive binding assay.The ability of VEGF165 (filled triangles: ▾), wildtype VEGF-C (filledcircles: ●), and three VEGF-C mutants [VEGF-C R226,227S (open boxes: □);VEGF-C ΔNΔCHis (open circles: ◯); and VEGF-C ΔNΔCH is C156S (opentriangles: Δ)] to compete with ¹²⁵I-VEGF-CΔNΔCHis for binding to VEGFR-2and VEGFR-3 is shown.

FIG. 5 depicts the amino acid sequences of human (SEQ ID NO: 8), murine(SEQ ID NO: 11), and quail (SEQ ID NO: 13) VEGF-C polypeptides, alignedto show similarity. Residues conserved in all three species are depictedin bold.

FIGS. 6A-C depict electrophoretic fractionations of the various forms ofrecombinant VEGF-C produced by transfected 293 EBNA cells. FIG. 6Bdepicts the electrophoretic fractionation, under non-reducingconditions, of polypeptides produced from mock (M) transfected cells,cells transfected with wild type (wt) VEGF-C cDNA, and cells transfectedwith a cDNA encoding the VEGF-C mutant VEGF-C-R102S. Each of the bandsidentified in FIG. 6B was excised and electrophoretically fractionatedin a separate lane under reducing conditions. Fractionation of bandscorresponding to wt VEGF-C are depicted in FIG. 6A; fractionation ofbands corresponding to the R102S mutant are depicted in FIG. 6C.

FIGS. 7A-B depict the forms and sizes of wild type and mutantrecombinant VEGF-Cs, as revealed by non-reducing gel electrophoresis.FIG. 7A shows the VEGF-C forms secreted into the media; FIG. 7B showsthe VEGF-C forms retained by the cells. Mock (M) transfected cellsserved as a control.

FIGS. 8A-B present a comparison of the pattern of immunoprecipitated,labeled VEGF-C forms using antisera 882 and antisera 905. Adjacent lanescontain immunoprecipitates that were (lanes marked +) or were not (lanesmarked −) subjected to reduction and alkylation.

FIG. 9 is a schematic model of the proteolytic processing of VEGF-C. Theregions of the VEGF-C polypeptide are depicted as follows: signalsequence=dark shaded box; VEGF-homology domain=medium shaded box;N-terminal and C-terminal propeptides=dotted and open boxes,respectively. Conserved cysteine residues in the VEGF-homology domainare depicted with dots (for clarity, cysteine residues in the C-terminalpropeptide are not marked). Putative sites of N-linked glycosylation areshown with Y symbols. Numbers indicate approximate molecular mass (kDa)of the corresponding polypeptide as measured by SDS-PAGE in reducingconditions. Disulfide bonds are marked as —S—S—; non-covalent bonds aredepicted as dotted lines. A question mark indicates the presence of apossible non-covalent bond. The proteolytic generation of a smallfraction of disulfide-linked 21 kDa forms is not indicated in thefigure. Several intermediate forms also are omitted to simplify thescheme. Particularly, only one precursor polypeptide is cleavedinitially. The figure is not intended to suggest that other intermediateforms, for example 21 kDa+31 kDa, 31 kDa+31 kDa+29 kDa, do not exist.

FIG. 10 presents a comparison of the human and mouse VEGF-C amino acidsequences. The amino acid sequence of mouse VEGF-C is presented on thetop line and differences in the human sequence are marked below it. Anarrow indicates the putative cleavage site for the signal peptidase;BR3P motifs, as well as a CR/SC motif, are boxed; and conserved cysteineresidues are marked in bold above the sequence. Arginine residue 158 isalso marked in bold. The numbering refers to mouse VEGF-C residues.

FIGS. 11A and 11B depict the genomic structure of the human (11A) andmurine (11B) VEGF-C genes. Sequences of exon-intron junctions aredepicted together with exon and intron lengths. Intron sequences aredepicted in lower case letters. Nucleotides of the open reading frameobserved in VEGF-C cDNAs are indicated as upper case letters in triplets(corresponding to the codons encoded at the junctions).

FIG. 12 depicts the exon-intron organization of the human VEGF-C gene.Seven exons are depicted as open boxes, with exon size depicted in basepairs. Introns are depicted as lines, with intron size (base pairs)depicted above the lines. 5′ and 3′ untranslated sequences of a putative2.4 kb mature mRNA are depicted as shaded boxes. The location of genomicclones used to characterize the VEGF-C gene are depicted below the mapof the gene.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is the isolation of a novel vascular endothelial growthfactor and the cloning of a DNA encoding this novel growth factor from acDNA library prepared from the human prostatic adenocarcinoma cell linePC-3. The isolated cDNA encodes a protein which is proteolyticallyprocessed and secreted to cell culture medium. The processing isdescribed in detail below. The secreted protein, designated VEGF-C,binds to the extracellular domain and induces tyrosineautophosphorylation of both Flt4 (VEGFR-3) and KDR/flk-1 (VEGFR-2). Incontrast, neither VEGF nor PlGF show high affinity binding to VEGFR-3 orinduced its autophosphorylation. VEGF-C also stimulates the migration ofendothelial cells in collagen gel and induces vascular permeability invivo. In transgenic mice, VEGF-C induces proliferation of the lymphaticendothelium and an causes an increase in neutrophilic granulocytes.Based on studies of VEGF-C variants and analogs and studies of VEGFprecursors, it is anticipated that one or more VEGF-C precursors (thelargest putative native VEGF-C precursor, excluding signal peptide,having the complete amino acid sequence from residue 32 to residue 419of SEQ ID NO: 8) is capable of stimulating VEGFR-3.

In addition to providing a cDNA sequence encoding prepro-VEGF-C, thepresent application also provides significant guidance concerningportions of the VEGF-C amino acid sequence which are necessary forbiological activity and portions (of one or more amino acids) which,when altered, will modulate (up-regulate or inhibit) VEGF-C biologicalactivities. Such alterations are readily achieved through recombinantDNA and protein techniques, such as site-directed mutagenesis of aVEGF-C encoding cDNA and recombinant expression of the resultantmodified cDNA. The skilled artisan also understands that, in recombinantproduction of proteins, additional sequence may be expressed along witha sequence encoding a polypeptide having a desired biological activity,while retaining a desired biological activity of the protein. Forexample, additional amino acids may be added at the amino terminus, atthe carboxy-terminus, or as an insertion into the polypeptide sequence.Similarly, deletion variants of a protein with a desired biologicalactivity can be recombinantly expressed that lack certain residues ofthe endogenous/natural protein, while retaining a desired biologicalactivity. Moreover, it is well-known that recombinant protein variantsmay be produced having conservative amino acid replacements (includingbut not limited to substitution of one or more amino acids for otheramino acids having similar chemical side-chains (acidic, basic,aliphatic, aliphatic hydroxyl, aromatic, amide, etc.)) which do noteliminate the desired biological activity of the protein. Accordingly,it is anticipated that such alterations of VEGF-C are VEGF-C equivalentswithin the scope of the invention.

As set forth in greater detail below, the putative prepro-VEGF-C has adeduced molecular mass of 46,883; a putative prepro-VEGF-C processingintermediate has an observed molecular weight of about 32 kD; and matureVEGF-C isolated from conditioned media has a molecular weight of about23 kD as assessed by SDS-PAGE under reducing conditions. A major part ofthe difference in the observed molecular mass of the purified andrecombinant VEGF-C and the deduced molecular mass of the prepro-VEGF-Cencoded by the VEGF-C open reading frame (ORF) is attributable toproteolytic removal of sequences at the amino-terminal andcarboxyl-terminal regions of the prepro-VEGF-C polypeptide.Extrapolation from studies of the structure of PDGF (Heldin et al.,Growth Factors, 8:245-52 (1993)) suggests that the region critical forreceptor binding and activation by VEGF-C is contained within aminoacids residues 104-213, which are found in the secreted form of theVEGF-C protein (i.e., the form lacking the putative prepro leadersequence and some carboxyterminal sequences). The 23 kD polypeptidebinding VEGFR-3 corresponds to a VEGF-homologous domain of VEGF-C. Afterbiosynthesis, the nascent VEGF-C polypeptide may be glycosylated atthree putative N-linked glycosylation sites identified in the deducedVEGF-C amino acid sequence. Polypeptides containing modifications, suchas N-linked glycosylations, are intended as aspects of the invention.

The carboxyl terminal amino acid sequences, which increase the length ofthe VEGF-C polypeptide in comparison with other ligands of this family,show a pattern of spacing of cysteine residues reminiscent of theBalbiani ring 3 protein (BR3P) sequence (Dignam et al., Gene, 88:133-40(1990); Paulsson et al., J. Mol. Biol., 211:331-49 (1990)). This novelC-terminal silk protein-like structural motif of VEGF-C may fold into anindependent domain, which is cleaved off after biosynthesis.Interestingly, at least one cysteine motif of the BR3P type is alsofound in the carboxyl terminus of VEGF. As explained in detail below,putative precursors and putative fully-processed VEGF-C were bothdetected in the cell culture media, suggesting cleavage by cellularproteases. The determination of amino-terminal and carboxy-terminalsequences of VEGF-C isolates was performed to identify the proteolyticprocessing sites. Antibodies generated against different parts of thepro-VEGF-C molecule were used to determine the precursor-productrelationship and ratio, their cellular distribution, and the kinetics ofprocessing and secretion.

VEGF-C has a conserved pattern of eight cysteine residues, which mayparticipate in the formation of intra- and interchain disulfide bonds,creating an antiparallel, dimeric, biologically active molecule, similarto PDGF. Mutational analysis of the cysteine residues involved in theinterchain disulfide bridges has shown that, in contrast to PDGF, VEGFdimers need to be held together by these covalent interactions in orderto maintain biological activity. Disulfide linking of the VEGF-Cpolypeptide chains was evident in the analysis of VEGF-C in nonreducingconditions, although recombinant protein also contained “fullyprocessed” ligand-active VEGF-C forms which lacked disulfide bondsbetween the polypeptides. (See FIG. 9.)

VEGFR-3, which distinguishes between VEGF and VEGF-C, is closely relatedin structure to VEGFR-1 and VEGFR-2. Finnerty et al., Oncogene,8:2293-98 (1993); Galland et al., Oncogene, 8:1233-40 (1993); Pajusolaet al., Cancer Res., 52:5738-43 (1992). Besides VEGFR-3, VEGFR-2tyrosine kinase also is activated in response to VEGF-C. VEGFR-2mediated signals cause striking changes in the morphology, actinreorganization and membrane ruffling of porcine aortic endothelial cellsover-expressing this receptor. In these cells, VEGFR-2 also mediatedligand-induced chemotaxis and mitogenicity. Waltenberger et al., J.Biol. Chem., 269:26988-95 (1994). Similarly, the receptor chimeraCSF-1R/VEGFR-3 was mitogenic when ectopically expressed in NIH 3T3fibroblastic cells, but not in porcine aortic endothelial cells(Pajusola et al., 1994). Consistent with such results, the bovinecapillary endothelial (BCE) cells, which express VEGFR-2 mRNA but verylittle or no VEGFR-1 or VEGFR-3 mRNAs, showed enhanced migration whenstimulated with VEGF-C. Light microscopy of the BCE cell cultures incollagen gel also suggested that VEGF-C stimulated the proliferation ofthese cells. The data thus indicate that the VEGF family of ligands andreceptors show a great specificity in their signaling, which may becell-type-dependent.

The expression pattern of the VEGFR-3 (Kaipainen et al., Proc. Natl.Acad. Sci. (USA), 92:3566-70 (1995)) suggests that VEGF-C may functionin the formation of the venous and lymphatic vascular systems duringembryogenesis. Constitutive expression of VEGF-C in adult tissues shownherein further suggests that this gene product also is involved in themaintenance of the differentiated functions of the lymphatic and certainvenous endothelia where VEGFR-3 is expressed (Kaipainen et al., 1995).Lymphatic capillaries do not have well-formed basal laminae and aninteresting possibility exists that the silk-like BR3P motif is involvedin producing a supramolecular structure which could regulate theavailability of VEGF-C in tissues. However, as shown here, VEGF-C alsoactivates VEGFR-2, which is abundant in proliferating endothelial cellsof vascular sprouts and branching vessels of embryonic tissues, but notso abundant in adult tissues. Millauer et al., Nature, 367:576-78(1993). These data have suggested that VEGFR-2 is a major regulator ofvasculogenesis and angiogenesis. VEGF-C may thus have a unique effect onlymphatic endothelium and a more redundant function, shared with VEGF,in angiogenesis and possibly in regulating the permeability of severaltypes of endothelia. Because VEGF-C stimulates VEGFR-2 and promotesendothelial migration, VEGF-C may be useful as an inducer ofangiogenesis of blood and lymphatic vessels in wound healing, in tissuetransplantation, in eye diseases, and in the formation of collateralvessels around arterial stenoses and into injured tissues afterinfarction.

Previously-identified growth factors that promote angiogenesis includethe fibroblast growth factors, hepatocyte growth factor/scatter factor,PDGF and TGF-α. (See e.g., Folkman, Nature Med., 1:27-31 (1995); Frieselet al., FASEB J., 9:919-25 (1995); Mustonen et al., J. Cell. Biol.,129:895-98 (1995). However, VEGF has been the only growth factorrelatively specific for endothelial cells. The newly identified factorsVEGF-B [Olofsson et al., Proc. Natl. Acad. Sci., 93:2578-81 (1996)] andVEGF-C thus increase our understanding of the complexity of the specificand redundant positive signals for endothelial cells involved invasculogenesis, angiogenesis, permeability, and perhaps also otherendothelial functions. Expression studies using Northern blotting showabundant VEGF-C expression in heart and skeletal muscle; other tissues,such as placenta, ovary, small intestine, thyroid gland, kidney,prostate, spleen, testis and large intestine also express this gene.Whereas PlGF is predominantly expressed in the placenta, the expressionpatterns of VEGF, VEGF-B and VEGF-C overlap in many tissues, whichsuggests that members of the VEGF family may form heterodimers andinteract to exert their physiological functions.

Targeted mutagenesis leading to inactivation of the VEGF receptor lociin the mouse genome has shown that VEGFR-1 is necessary for the properorganization of endothelial cells forming the vascular endothelium,while VEGFR-2 is necessary for the generation of both endothelial andhematopoietic cells. This suggests that the four genes of the VEGFfamily can be targets for mutations leading to vascular malformations orcardiovascular diseases.

The following Examples illustrate preferred embodiments of theinvention, wherein the isolation, characterization, and function ofVEGF-C, VEGF-C variants and analogs, VEGF-C-encoding nucleic acids, andanti-VEGF-C antibodies according to the invention are shown.

Example 1 Production of pLTRFlt41 Expression Vector

The identification and isolation of two forms of Flt4 receptor tyrosinekinase (VEGFR-3) cDNA (Flt4 short form (Flt4s), Genbank Accession No.X68203, SEQ ID NO: 1; and Flt4 long form, (Flt41), Genbank AccessionNos. X68203 and S66407, SEQ ID NO: 2) was reported in U.S. patentapplication Ser. No. 08/340,011, filed Nov. 14, 1994, incorporated byreference herein. An Flt4 expression vector designated pLTRFlt41(encoding the long form of Flt4) was constructed using the pLTRpolyexpression vector reported in Mäkelä et al., Gene, 118: 293-294 (1992)(Genbank accession number X60280, SEQ ID NO: 3) and the Flt4 cDNAs, inthe manner described in commonly-owned PCT patent applicationPCT/FI96/00427, filed Aug. 1, 1996, published as PCT publication No. WO97/05250 on 13 Feb. 1997, and commonly-owned U.S. patent applicationSer. No. 08/671,573, filed Jun. 28, 1996; 08/601,132, filed Feb. 14,1996; 08/585,895, filed Jan. 12, 1996; and 08/510,133, filed Aug. 1,1995, all of which are incorporated by reference in their entirety.

Example 2 Production and Analysis of Flt41 Transfected Cells

NIH 3T3 cells (60% confluent) were co-transfected with 5 micrograms ofthe pLTRFlt41 construct and 0.25 micrograms of the pSV2neo vectorcontaining the neomycin phosphotransferase gene (Southern et al., J.Mol. Appl. Genet., 1:327 (1982)), using the DOTAP liposome-basedtransfection reagents (Boehringer-Mannheim, Mannheim, Germany). One dayafter transfection, the cells were transferred into selection mediacontaining 0.5 mg/ml geneticin (GIBCO, Grand Island, N.Y.). Colonies ofgeneticin-resistant cells were isolated and analyzed for expression ofthe Flt4 proteins. Cells were lysed in boiling lysis buffer containing3.3% SDS and 125 mM Tris, pH 6.8. Protein concentrations of the sampleswere measured by the BCA method (Pierce, Rockford, Ill.). About 50micrograms of protein from each lysate were analyzed for the presence ofFlt4 by 6% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andimmunoblotting using antisera against the carboxyl terminus of Flt4.Signals on Western blots were revealed using the ECL method (Amersham).

For production of anti-Flt4 antiserum, the Flt4 cDNA fragment encodingthe 40 carboxy-terminal amino acid residues of the Flt4 short form:NH2-PMTPTTYKG SVDNQTDSGM VLASEEFEQI ESRHRQESGFR-COOH (SEQ ID NO: 4) wascloned as a 657 by EcoRI-fragment into the pGEX-1λT bacterial expressionvector (Pharmacia LKB, Inc., Uppsala, Sweden) in frame with theglutathione-S-transferase coding region. The resultant GST-Flt4S fusionprotein was produced in E. coli and purified by affinity chromatographyusing a glutathione-Sepharose 4B column. The purified protein waslyophilized, dissolved in phosphate-buffered saline (PBS), mixed withFreund's adjuvant and used for immunization of rabbits at bi-weeklyintervals using methods standard in the art (Harlow et al., Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988)).Antisera were used, after the fourth booster immunization, forimmunoprecipitation of Flt4 from transfected cells. Cell clonesexpressing Flt4 were also used for ligand stimulation analysis.

Example 3 Construction of a Flt4 EC Baculovirus Vector and Expressionand Purification of its Product

Using the pVTBac plasmid described in Tessier et al., Gene 98:177-183(1991), and the Flt4 cDNAs described in Example 1, a baculovirusexpression vector was constructed to facilitate expression of theextracellular domain of Flt4 (Flt4 EC), as described in commonly-ownedPCT patent application PCT/FI96/00427, filed Aug. 1, 1996, published asPCT publication No. WO 97/05250 on 13 Feb. 1997, and commonly-owned U.S.patent application Ser. No. 08/671,573, filed Jun. 28, 1996; 08/601,132,filed Feb. 14, 1996; 08/585,895, filed Jan. 12, 1996; and 08/510,133,filed Aug. 1, 1995, all of which are incorporated by reference herein. Anucleotide sequence encoding a 6×His tag was operatively connected tothe Flt4 EC coding sequence to facilitate purification.

The Flt4EC construct was transfected together with baculovirus genomicDNA into SF-9 cells by lipofection. Recombinant virus was purified,amplified and used for infection of High-Five cells (Invitrogen, SanDiego, Calif.) using methods standard in the art. The Flt4 extracellulardomain (Flt4EC) was purified from the culture medium of the infectedHigh-Five cells using Ni-NTA affinity chromatography according tomanufacturer's instructions (Qiagen) for binding and elution of the6×His tag encoded in the COOH-terminus of the recombinant Flt4extracellular domain.

Example 4 Isolation of an Flt4 Ligand from Conditioned Media

A human Flt4 ligand according to the invention was isolated from mediaconditioned by a PC-3 prostatic adenocarcinoma cell line (ATCC CRL 1435)in serum-free Ham's F-12 Nutrient mixture (GIBCO) (containing 7% fetalcalf serum (FCS)). Cells were reseeded and grown in this medium, whichwas subsequently changed to serum-free medium. The preparation of theconditioned media, and the identification of a component therein whichstimulated Flt4 tyrosine phosphorylation, are described in detail incommonly-owned PCT patent application PCT/FI96/00427, filed Aug. 1,1996, and commonly-owned U.S. patent application Ser. No. 08/671,573,filed Jun. 28, 1996; 08/601,132, filed Feb. 14, 1996; 08/585,895, filedJan. 12, 1996; 08/510,133, filed Aug. 1, 1995; and 08/340,011, filedNov. 14, 1994, all of which are incorporated by reference herein intheir entirety. The ability of the conditioned medium to stimulate Flt4phosyphorylation was considerably increased when the PC-3 conditionedmedium was concentrated four-fold using a Centricon-10 concentrator(Amicon). Pretreatment of the concentrated PC-3 conditioned medium with50 microliters of Flt4 extracellular domain coupled to CNBr-activatedsepharose CL-4B (Pharmacia; about 1 mg of Flt4EC domain/ml sepharoseresin) completely abolished Flt4 tyrosine phosphorylation. Similarpretreatment of the conditioned medium with unsubstituted sepharoseCL-4B did not affect stimulatory activity. Also, the flow throughobtained after concentration, which contained proteins of less than10,000 molecular weight, did not stimulate Flt4 phosphorylation.

In another experiment, a comparison of Flt4 autophosphorylation intransformed NIH 3T3 cells expressing LTRFlt41 was conducted, usingunconditioned medium, medium from PC-3 cells expressing the Flt4 ligand,or unconditioned medium containing either 50 ng/ml of VEGF165 or 50ng/ml of PlGF-1. The cells were lysed, immunoprecipitated usinganti-Flt4 antiserum and analyzed by Western blotting usinganti-phosphotyrosine antibodies. Only the PC-3 conditioned mediumexpressing the Flt4 ligand (lane Flt-4L) stimulated Flt4autophosphorylation.

These experiments showed that PC-3 cells produce a ligand which binds tothe extracellular domain of Flt4 and activates this receptor.

Example 5 Purification of the Flt4 Ligand

The ligand expressed by human PC-3 cells as characterized in Example 4was purified and isolated using a recombinantly-produced Flt4extracellular domain (Flt4EC) in affinity chromatography.

Two harvests of serum-free conditioned medium, comprising a total of 8liters, were collected from 500 confluent 15 cm diameter culture dishescontaining confluent layers of PC-3 cells. The conditioned medium wasclarified by centrifugation at 10,000×g and concentrated 80-fold usingan Ultrasette Tangential Flow Device (Filtron, Northborough, Mass.) witha 10 kD cutoff Omega Ultrafiltration membrane according to themanufacturer's instructions. Recombinant Flt4 extracellular domain wasexpressed in a recombinant baculovirus cell system and purified byaffinity chromatography on Ni-agarose (Ni-NTA affinity column obtainedfrom Qiagen). The purified extracellular domain was coupled toCNBr-activated Sepharose CL-4B at a concentration of 5 mg/ml and used asan affinity matrix for ligand affinity chromatography.

Concentrated conditioned medium was incubated with 2 ml of therecombinant Flt4 extracellular domain-Sepharose affinity matrix in arolling tube at room temperature for 3 hours. All subsequentpurification steps were at +4° C. The affinity matrix was thentransferred to a column with an inner diameter of 15 mm and washedsuccessively with 100 ml of PBS and 50 ml of 10 mM Na-phosphate buffer(pH 6.8). Bound material was eluted step-wise with 100 mM glycine-HCl,successive 6 ml elutions having pHs of 4.0, 2.4, and 1.9. Several 2 mlfractions of the eluate were collected in tubes containing 0.5 ml 1 MNa-phosphate (pH 8.0). Fractions were mixed immediately and dialyzed in1 mM Tris-HCl (pH 7.5). Aliquots of 75 μl each were analyzed for theirability to stimulate tyrosine phosphorylation of Flt4. Theultrafiltrate, 100 μl aliquots of the concentrated conditioned mediumbefore and after ligand affinity chromatography, as well as 15-foldconcentrated fractions of material released from the Flt4 extracellulardomain-Sepharose matrix during the washings were also analyzed for theirability to stimulate Flt4 tyrosine phosphorylation.

The concentrated conditioned medium induced prominent tyrosinephosphorylation of Flt4 in transfected NIH 3T3 cells over-expressingFlt4. This activity was not observed in conditioned medium taken aftermedium was exposed to the Flt4 Sepharose affinity matrix. Thespecifically-bound Flt4-stimulating material was retained on theaffinity matrix after washing in PBS, 10 mM Na-phosphate buffer (pH6.8), and at pH 4.0. It was eluted in the first two 2 ml aliquots at pH2.4. A further decrease of the pH of the elution buffer did not causerelease of additional Flt4-stimulating material. No Flt4 phosphorylationwas observed in a control wherein Flt4-expressing cells were treatedwith unconditioned medium; similarly, no phosphorylation was observedfollowing treatment of Flt4-expressing cells with the ultrafiltratefraction of conditioned medium containing polypeptides of less than 10kD molecular weight.

Small aliquots of the chromatographic fractions were concentrated in aSpeedVac concentrator (Savant, Farmingdale, N.Y.) and subjected toSDS-PAGE under reducing conditions with subsequent silver staining ofthe gel, a standard technique in the art. The major polypeptide, havinga molecular weight of approximately 23 kD (reducing conditions), wasdetected in the fractions containing Flt4 stimulating activity. Thatpolypeptide was not found in the other chromatographic fractions. On theother hand, besides these bands and a very faint band having a 32 kDmobility, all other components detected in the two active fractions werealso distributed in the starting material and in small amounts in theother washing and eluting steps after their concentration. Similarresults were obtained in three independent affinity purifications,indicating that the 23 kD polypeptide binds with high affinity to Flt4and induces tyrosine phosphorylation of Flt4.

Fractions containing the 23 kD polypeptide were combined, dried in aSpeedVac concentrator and subjected to SDS-PAGE in a 12.5% gel. Theproteins from the gel were then electroblotted to Immobilon-P(PVDF)transfer membrane (Millipore, Marlborough, Mass.) and visualized bystaining of the blot with Coomassie Blue R-250. The region containingonly the stained 23 kD band was cut from the blot and subjected toN-terminal amino acid sequence analysis in a Prosite Protein SequencingSystem (Applied Biosystems, Foster City, Calif.). The data were analyzedusing a 610A Data Analysis System (Applied Biosystems). Analysisrevealed a single N-terminal sequence of NH₂-XEETIKFAAA-HYNTEILK-COOH(SEQ ID NO: 5).

Example 6 Construction of PC-3 Cell cDNA Library in a EukaryoticExpression Vector

Human poly(A)⁺ RNA was isolated from five 15 cm diameter dishes ofconfluent PC-3 cells by a single step method using oligo(dT) (Type III,Collaborative Biomedical Products, Becton-Dickinson Labware, Bedford,Mass.) cellulose affinity chromatography (Sambrook et al., 1989). Theyield was 70 micrograms. Six micrograms of the Poly(A)⁺RNA were used toprepare an oligo(dT)-primed cDNA library in the mammalian expressionvector pcDNA I and the Librarian kit of Invitrogen according to theinstructions included in the kit. The library was estimated to containabout 10⁶ independent recombinants with an average insert size ofapproximately 1.8 kb.

Examples 7-9 Amplification of a cDNA Encoding the Flt4 Ligand AminoTerminus

The procedures used to isolate a cDNA encoding the Flt4 ligand aredescribed in detail in commonly-owned PCT patent applicationPCT/FI96/00427, filed Aug. 1, 1996, and commonly-owned U.S. patentapplication Ser. No. 08/671,573, filed Jun. 28, 1996; 08/601,132, filedFeb. 14, 1996; 08/585,895, filed Jan. 12, 1996; and 08/510,133, filedAug. 1, 1995, all of which are incorporated by reference herein.Initially, degenerate oligonucleotides were designed based on theN-terminal amino acid sequence of the isolated human Flt4 ligand (seeExample 5) and were used as primers in a polymerase chain reaction (PCR)to amplify a partial cDNA encoding the (fully-processed) Flt4 ligandamino terminus from the PC-3 cDNA library. The amplified cDNA fragmentwas cloned into a pCR II vector (Invitrogen) using the TA cloning kit(Invitrogen) and sequenced using the radioactive dideoxynucleotidesequencing method of Sanger. Six clones were analyzed and all six clonescontained the sequence encoding the expected peptide (amino acidresidues 104-120 of the Flt4 ligand precursor, SEQ ID NO: 8). Nucleotidesequence spanning the region from the third nucleotide of codon 6 to thethird nucleotide of codon 13 (the extension region between the PCRprimers) was identical in all six clones and thus represented anamplified product from the unique sequence encoding part of the aminoterminus of the Flt4 ligand.

Based on the unique nucleotide sequence encoding the N-terminus of theisolated human Flt4 ligand, two pairs of nested primers were designed toamplify, in two nested PCR reactions, the complete 5′-end of thecorresponding cDNAs from one microgram of DNA of the above-describedPC-3 cDNA library. One major product of about 220 by and three minorproducts of about 270 bp, 150 bp, and 100 by were obtained.

The amplified fragment of approximately 220 by was excised from anagarose gel, cloned into a pCR11 vector using the TA cloning kit, andsequenced. Three recombinant clones were analyzed and they contained thesequence 5′-TCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGGTGGAATTCGACGAACTCATGACTGTACTCTACCCAGAATATTGGAAAATGTACAAGTGTCAGCTAAGGCAAGGAGGCTGGCAACATAACAGAGAACAGGCCAACCTCAACTCAAGGACAGAAGAGACTATAAAATTCGCTGCAG CACACTACAAC-3′ (SEQID NO: 6). The beginning of the sequence represents the vector and theunderlined sequence represents the amplified product of the 5′-end ofthe cDNA insert.

Based upon the amplified 5′-sequence of the clones encoding the aminoterminus of the 23 kD human Flt4 ligand, two pairs of non-overlappingnested primers were designed to amplify the 3′-portion of theFlt4-ligand-encoding cDNA clones via PCR. Two DNA fragments wereobtained, having sizes of 1350 by and 570 bp. Those fragments werecloned into a pCRII vector and the inserts of the clones were sequenced.Both of these fragments were found to contain sequences encoding anamino acid sequence homologous to the VEGF sequence.

Example 10 Screening the PC-3 Cell cDNA Library Using the 5′ PCRFragment of Flt4 Ligand cDNA

A 153 by fragment encoding the 5′ end of the Flt4 ligand was labeledwith [³²P]-dCTP using the Klenow fragment of E. coli DNA polymerase I(Boehringer Mannheim). That fragment was used as a probe forhybridization screening of the amplified PC-3 cell cDNA library.

Filter replicas of the library were hybridized with the radioactivelylabeled probe at 42° C. for 20 hours in a solution containing 50%formamide, 5×SSPE, 5×Denhardt's solution, 0.1% SDS and 0.1 mg/mldenatured salmon sperm DNA. Filters were washed twice in 1×SSC, 0.1% SDSfor 30 minutes at room temperature, then twice for 30 minutes at 65° C.and exposed overnight.

On the basis of autoradiography, 10 positive recombinant bacterialcolonies hybridizing with the probe were chosen from the library.Plasmid DNA was purified from these colonies and analyzed by EcoRI andNotI digestion and agarose gel electrophoresis followed by ethidiumbromide staining. The ten plasmid clones were divided into three groupson the basis of the presence of insert sizes of approximately 1.7, 1.9and 2.1 kb, respectively. Inserts of plasmids from each group weresequenced using the T7 oligonucleotide as a primer and walking primersfor subsequent sequencing reactions.

Sequence analysis showed that all clones contain the open reading frameencoding the NH2-terminal sequence of the 23 kD human Flt4 ligand.Dideoxy sequencing was continued using walking primers in the downstreamdirection. A complete human cDNA sequence and deduced amino acidsequence from a 2 kb clone is set forth in SEQ ID NOs: 7 and 8,respectively. A putative cleavage site of a “prepro” leader sequence islocated between residues 102 and 103 of SEQ ID NO: 8. When compared withsequences in the GenBank Database, the predicted protein product of thisreading frame was found to include a region homologous with thepredicted amino acid sequences of the PDGFNEGF family of growth factors,as shown in FIG. 2.

Plasmid pFLT4-L, containing the 2.1 kb human cDNA clone in pcDNAIvector, has, been deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 as accession number 97231.

Example 11 Stimulation of Flt4 Autophosphorylation by the ProteinProduct of the Flt4 Ligand Vector

The 2.1 kb human cDNA insert of plasmid pFlt4-L, which contains the openreading frame encoding the sequence shown in SEQ ID NOs: 7 and 8; humanprepro-VEGF-C, see below), was cut out from the pcDNAI vector usingHindIII and NotI restriction enzymes, isolated from a preparativeagarose gel, and ligated to the corresponding sites in the pREP7expression vector (Invitrogen). The pREP7 vector containing the pFlt4-Linsert was transfected into 293-EBNA cells (Invitrogen) using thecalcium phosphate transfection method (Sambrook et al., 1989). About 48hours after transfection, the medium of the transfected cells waschanged to DMEM medium lacking fetal calf serum and incubated for 36hours. The conditioned medium was then collected, centrifuged at 5000×gfor 20 minutes, the supernatant was concentrated 5-fold using Centriprep10 (Amicon) and used to stimulate NIH 3T3 cells expressing LTRFlt41 (theFlt4 receptor), as in Example 4. The cells were lysed,immunoprecipitated using anti-Flt4 antiserum and analyzed by Westernblotting using anti-phosphotyrosine antibodies.

The conditioned medium from two different dishes of the transfectedcells stimulated Flt4 autophosphorylation in comparison with the mediumfrom mock-transfected cells, which gave only background levels ofphosphorylation of the Flt4 receptor. When the concentrated conditionedmedium was pre-absorbed with 20 microliters of a slurry of Flt4EC domaincoupled to Sepharose (see example 4), no phosphorylation was obtained,showing that the activity responsible for Flt4 autophosphorylation wasindeed the Flt4 ligand. Thus, these results demonstrate that anexpression vector having an approximately 2.1 kb insert and containingan open reading frame as shown in SEQ ID. NO: 7 is expressed as abiologically active Flt4 ligand (VEGF-C) in transfected cells. Thesequence encoded by that open reading frame is shown in SEQ ID NO: 8.

The deduced molecular weight of a polypeptide consisting of the completeamino acid sequence in SEQ ID NO: 8 (residues 1 to 419) is 46,883. Thededuced molecular weight of a polypeptide consisting of amino acidresidues 103 to 419 of SEQ ID NO: 8 is 35,881. The Flt4 ligand purifiedfrom PC-3 cultures had an observed molecular weight of about 23 kD asassessed by SDS-PAGE under reducing conditions. Thus, it appeared thatthe Flt4 ligand mRNA was translated into a precursor polypeptide, fromwhich the mature ligand was derived by proteolytic cleavage. Also, theFlt4 ligand may be glycosylated at three putative N-linked glycosylationsites conforming to the consensus which can be identified in the deducedFlt4 ligand amino acid sequence (N-residues underlined in FIG. 2).

The carboxyl terminal amino acid sequences, which increase the predictedmolecular weight of the Flt4 ligand subunit in comparison with otherligands of this family, show a pattern of spacing of cysteine residuesreminiscent of the Balbiani ring 3 protein (BR3P) sequence (Dignam etal., Gene, 88:133-140 (1990)). Such a sequence may encode anindependently folded domain present in a Flt4 ligand precursor and itmay be involved, for example, in the regulation of secretion,solubility, stability, cell surface localization or activity of the Flt4ligand. Interestingly, at least one cysteine motif of the BR3P type isalso found in the VEGF carboxy terminal amino acid sequences.

Thus, the Flt4 ligand mRNA appears first to be translated into aprecursor from the mRNA corresponding to the cDNA insert of plasmidFLT4-L, from which the mature ligand is derived by proteolytic cleavage.To define the mature Flt4 ligand polypeptide, one first expresses thecDNA clone (which is deposited in the pcDNAI expression vector) incells, such as COS cells. One uses antibodies generated against encodedpolypeptides, fragments thereof, or bacterial Flt4 fusion proteins, suchas a GST-fusion protein, to raise antibodies against the VEGF-homologousdomain and the amino- and carboxyl-terminal propeptides of Flt4 ligand.One then follows the biosynthesis and processing of the Flt4 ligand inthe transfected cells by pulse-chase analysis using radioactive cysteinefor labeling of the cells, immunoprecipitation, and gel electrophoresis.Using antibodies against the three domains of the product encoded by thecDNA insert of plasmid FLT4-L, material for radioactive ornonradioactive amino-terminal sequence analysis is isolated. Thedetermination of the amino-terminal sequence of the mature VEGF-Cpolypeptide allows for identification of the amino-terminal proteolyticprocessing site. The determination of the amino-terminal sequence of thecarboxyl-terminal propeptide will give the carboxyl-terminal processingsite. This is confirmed by site-directed mutagenesis of the amino acidresidues adjacent to the cleavage sites, which would prevent thecleavage.

The Flt4 ligand is further characterizeable by progressive 3′ deletionsin the 3′ coding sequences of the Flt4 ligand precursor clone,introducing a stop codon resulting in carboxy-terminal truncations ofits protein product. The activities of such truncated forms are assayedby, for example, studying Flt4 autophosphorylation induced by thetruncated proteins when applied to cultures of cells, such as NIH 3T3cells expressing LTRFlt41. By extrapolation from studies of thestructure of the related platelet derived growth factor (PDGF, Heldin etal., Growth Factors, 8:245-252 (1993)) one determines that the regioncritical for receptor activation by the Flt4 ligand is contained withinthe first approximately 180 amino acid residues of the secreted VEGF-Cprotein lacking the putative 102 amino acid prepro leader (SEQ. ID NO:8, residues 103-282), and apparently within the first approximately 120amino acid residues (SEQ ID NO: 8, residues 103-223).

On the other hand, the difference between the molecular weights observedfor the purified ligand and deduced from the open reading frame of theFlt4 ligand clone may be due to the fact that the soluble ligand wasproduced from an alternatively spliced mRNA which would also be presentin the PC-3 cells, from which the isolated ligand was derived. Toisolate such alternative cDNA clones one uses cDNA fragments of thedeposited clone and PCR primers made according to the sequence providedas well as techniques standard in the art to isolate or amplifyalternative cDNAs from the PC-3 cell cDNA library. One may also amplifyusing reverse transcription (RT)-PCR directly from the PC-3 mRNA usingthe primers provided in the sequence of the cDNA insert of plasmidFLT4-L. Alternative cDNA sequences are determined from the resultingcDNA clones. One can also isolate genomic clones corresponding to theFlt4 ligand mRNA transcript from a human genomic DNA library usingmethods standard in the art and sequence such clones or their subclonedfragments to reveal the corresponding exons. Alternative exons can thenbe identified by a number of methods standard in the art, such asheteroduplex analysis of cDNA and genomic DNA, which are subsequentlycharacterized.

Example 12 Expression of the Gene Encoding VEGF-C in Human Tumor CellLines

Expression of transcripts corresponding to the Flt4 ligand (VEGF-C) wasanalyzed by hybridization of Northern blots containing isolated poly(A)⁺RNA from HT-1080 and PC-3 human tumor cell lines. The probe was theradioactively labeled insert of the 2.1 kb cDNA clone (pFlt4-L/VEGF-C,specific activity 10⁸-10⁹ cpm/mg of DNA). The blot was hybridizedovernight at 42° C. using 50% formamide, 5×SSPE buffer, 2% SDS,10×Denhardt's solution, 100 mg/ml salmon sperm DNA and 1×10⁶ cpm of thelabeled probe/ml. The blot was washed at room temperature for 2×30minutes in 2×SSC containing 0.05% SDS, and then for 2×20 minutes at 52°C. in 0.1×SSC containing 0.1% SDS. The blot was then exposed at −70° C.for three days using intensifying screens and Kodak XAR film. Both celllines expressed an Flt4 ligand mRNA of about 2.4 kb, as well as VEGF andVEGF-B mRNAs.

Example 13 VEGF-C Chains Are Proteolytically Processed afterBiosynthesis and Disulfide Linked

The predicted molecular mass of a secreted human VEGF-C polypeptide, asdeduced from the VEGF-C open reading frame, is 46,883 kD, suggestingthat VEGF-C mRNA may be first translated into a precursor, from whichthe observed ligands of 21/23 kD and 29/32 kD are derived by proteolyticcleavage.

This possibility was explored by metabolic labeling of 293 EBNA cellsexpressing VEGF-C. Initially, 293 EBNA cells were transfected with theVEGF-C cDNA construct. Expression products were labeled by the additionof 100 μCi/ml of Pro-mix™ L-[³⁵S] in vitro cell labeling mix((containing ³⁵S-methionine and ³⁵S-cysteine) Amersham, Buckinghamshire,England) to the culture medium devoid of cysteine and methionine. Aftertwo hours, the cell layers were washed twice with PBS and the medium wasthen replaced with DMEM-0.2% BSA. After 1, 3, 6, 12 and 24 hours ofsubsequent incubation, the culture medium was collected, clarified bycentrifugation, and concentrated, and human VEGF-C was bound to 30 μl ofa slurry of Flt4EC-Sepharose overnight at +4° C., followed by threewashes in PBS, two washes in 20 mM Tris-HCl (pH 7.5), alkylation,SDS-PAGE and autoradiography. Alkylation was carried out by treatment ofthe samples with 10 mM 1,4 Dithiothreitol (Boehringer-Mannheim,Mannheim, Germany) for one hour at 25° C., and subsequently with 30 mMiodoacetamide (Fluka, Buchs, Switzerland).

These experiments demonstrated that a putative precursor polypeptide of32 kD apparent molecular mass was bound to the Flt4EC affinity matrixfrom the conditioned medium of metabolically labeled cells transfectedwith the human VEGF-C expression vector, but not from mock transfectedcells. Increased amounts of a 23 kD receptor binding polypeptideaccumulated in the culture medium of VEGF-C transfected cells during asubsequent chase period of three hours, but not thereafter, suggestingthat the 23 kD form is produced by proteolytic processing, which isincomplete, at least in the transiently transfected cells. Subsequentexperiments showed that the 3210 VEGF-C form contains two componentsmigrating in the absence of alkylation as polypeptides of 29 and 32 kD(FIGS. 6-8).

In a related experiment, human VEGF-C isolated using Flt4EC-Sepharoseafter a 4 hour continuous metabolic labeling was analyzed bypolyacrylamide gel electrophoresis in nonreducing conditions. Highermolecular mass forms were observed under nonreducing conditions,suggesting that the VEGF-C polypeptides can form disulfide-linked dimersand/or multimers. Gel photographs depicting these experimental resultsare set forth in FIGS. 13A-B of PCT application PCT/FI96/00427(publication WO 97/05250) and FIGS. 3A-B of U.S. patent application Ser.No. 08/795,430, which are incorporated herein by reference.

Additional experiments have shown that higher molecular mass forms ofVEGF-C (about 58 kD and about 43 kD) are observed under reducingconditions as well. (See below and FIG. 6A.)

Example 14 Stimulation Of VEGFR-2 Autophosphorylation By VEGF-C

Conditioned medium (CM) from 293 EBNA cells transfected with the humanVEGF-C vector also was used to stimulate porcine aortic endothelial(PAE) cells expressing VEGFR-2 (KDR). Pajusola et al., Oncogene,9:3545-55 (1994); Waltenberger et al., J. Biol. Chem., 269:26988-26995(1994). The cells were lysed and immunoprecipitated usingVEGFR-2-specific antiserum (Waltenberger et al., 1994).

PAE-KDR cells (Waltenberger et al., 1994) were grown in Ham's F12medium-10% fetal calf serum (FCS). Confluent NIH 3T3-Flt4 cells orPAE-KDR cells were starved overnight in DMEM or Ham's F12 medium,respectively, supplemented with 0.2% bovine serum albumin (BSA), andthen incubated for 5 minutes with the analyzed media. Recombinant humanVEGF (R&D Systems) and PDGF-BB, functional as stimulating agents, wereused as controls. The cells were washed twice with ice-coldTris-Buffered Saline (TBS) containing 100 mM sodium orthovanadate andlysed in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride(PMSF), 0.1 U/ml aprotinin and 1 mM sodium orthovanadate. The lysateswere sonicated, clarified by centrifugation at 16,000×g for 20 minutesand incubated for 3-6 hours on ice with 3-5 μl of antisera specific forFlt4 (Pajusola et al., 1993), VEGFR-2 or PDGFR-β (Claesson-Welsh et al.,J. Biol. Chem., 264:1742-1747 (1989); Waltenberger et al., 1994).Immunoprecipitates were bound to protein A-Sepharose, washed three timeswith RIPA buffer containing 1 mM PMSF, 1 mM sodium orthovanadate, washedtwice with 10 mM Tris-HCl (pH 7.4), and subjected to SDS-PAGE using a 7%gel. Polypeptides were transferred to nitrocellulose by Western blottingand analyzed using PY20 phosphotyrosine-specific monoclonal antibodies(Transduction Laboratories) or receptor-specific antiserum and the ECLdetection method (Amersham Corp.).

PAE cells expressing VEGFR-2 were treated with 10- or 2-foldconcentrated medium from mock-transfected 293-EBNA cells, or with 2-, 5-or 10-fold concentrated medium from 293-EBNA cell cultures expressingthe recombinant VEGF-C. VEGFR-2 was immunoprecipitated with specificantibodies and analyzed by SDS-PAGE and Western blotting usingphosphotyrosine antibodies. For comparison, the treatments were alsocarried out with non-conditioned medium containing 50 ng/ml of purifiedrecombinant VEGF. Additional cells were also treated with VEGF-C- orVEGF-containing media pretreated with Flt4EC.

The results of this experiment were as follows. A basal level oftyrosine phosphorylation of VEGFR-2 was detected in cells stimulated byCM from the mock-transfected cells. A further concentration of thismedium resulted in only a slight enhancement of VEGFR-2 phosphorylation.CM containing recombinant VEGF-C stimulated tyrosine autophosphorylationof VEGFR-2 and the intensity of the autophosphorylated polypeptide bandwas increased upon concentration of the VEGF-C CM. Furthermore, thestimulating effect was abolished after pretreatment of the medium withthe Flt4EC affinity matrix. The maximal effect of VEGF-C in this assaywas comparable to the effect of recombinant VEGF added to unconditionedmedium at concentration of 50 ng/ml. Pretreatment of the mediumcontaining VEGF with Flt4EC did not abolish its stimulating effect onVEGFR-2. These results suggest that the VEGF-C expression vector encodesa ligand not only for Flt4 (VEGFR-3), but also for KDR/Flk-1 (VEGFR-2).

In order to further confirm that the stimulating effect of VEGF-C ontyrosine phosphorylation of VEGFR-3 and VEGFR-2 was receptor-specific,we analyzed the effect of VEGF-C on tyrosine phosphorylation of PDGFreceptor β (PDGFR-β) which is abundantly expressed on fibroblasticcells. PDGFR-β-expressing NIH 3T3 cells were treated withnon-conditioned medium, 5-fold concentrated CM from mock-transfected orVEGF-C-transfected cells, or with non-conditioned medium containing 50ng/ml of recombinant human PDGF-BB. Medium containing VEGF-C was alsopretreated with recombinant Flt4EC (lane 4). PDGFR-β wasimmunoprecipitated with specific antibodies and analyzed by SDS-PAGE andWestern blotting using phosphotyrosine antibodies with subsequentstripping and reprobing of the membrane with antibodies specific forPDGFR-β. A weak tyrosine phosphorylation of PDGFR-β was detected uponstimulation of Flt4-expressing NIH 3T3 cells with CM from themock-transfected cells. A similar low level of PDGFR-β phosphorylationwas observed when the cells were incubated with CM from the VEGF-Ctransfected cells, with or without prior treatment with FIt4EC. Incontrast, the addition of 50 ng/ml of PDGF-BB induced a prominenttyrosine autophosphorylation of PDGFR-β.

Example 15 VEGF-C Stimulates Endothelial Cell Migration in Collagen Gel

Conditioned media (CM) from cell cultures transfected with the VEGF-Cexpression vector was placed in a well made in collagen gel and used tostimulate the migration of bovine capillary endothelial (BCE) cells inthe three-dimensional collagen gel as follows.

BCE cells (Folkman et al., Proc. Natl. Acad. Sci. (USA), 76:5217-5221(1979)) were cultured as described in Pertovaara et al., J. Biol. Chem.,269:6271-74 (1994). The collagen gels were prepared by mixing type Icollagen stock solution (5 mg/ml in 1 mM HCl) with an equal volume of2×MEM and 2 volumes of MEM containing 10% newborn calf serum to give afinal collagen concentration of 1.25 mg/ml. The tissue culture plates (5cm diameter) were coated with about 1 mm thick layer of the solution,which was allowed to polymerize at 37° C. BCE cells were seeded on topof this layer. For the migration assays, the cells were allowed toattach inside a plastic ring (1 cm diameter) placed on top of the firstcollagen layer. After 30 minutes, the ring was removed and unattachedcells were rinsed away. A second layer of collagen and a layer of growthmedium (5% newborn calf serum (NCS)), solidified by 0.75% low meltingpoint agar (FMC BioProducts, Rockland, Me.), were added. A well (3 mmdiameter) was punched through all the layers on both sides of the cellspot at a distance of 4 mm, and the sample or control media werepipetted daily into the wells. Photomicrographs of the cells migratingout from the spot edge were taken after six days through an Olympus CK 2inverted microscope equipped with phase-contrast optics. The migratingcells were counted after nuclear staining with the fluorescent dyebisbenzimide (1 mg/ml, Hoechst 33258, Sigma).

The number of cells migrating at different distances from the originalarea of attachment towards wells containing media conditioned by thenon-transfected (control) or transfected (mock; VEGF-C; VEGF) cells weredetermined 6 days after addition of the media. The number of cellsmigrating out from the original ring of attachment was counted in fiveadjacent 0.5 mm×0.5 mm squares using a microscope ocular lens grid and10× magnification with a fluorescence microscope. Cells migratingfurther than 0.5 mm were counted in a similar way by moving the grid in0.5 mm steps. The experiments were carried out twice with similarresults. At each distance, VEGF-C-containing CM stimulated cellmigration more than medium conditioned by the non-transfected ormock-transfected cells but less than medium from cells transfected witha VEGF expression vector. Daily addition of 1 ng of FGF2 into the wellsresulted in the migration of approximately twice the number of cellswhen compared to the stimulation by CM from VEGF-transfected cells.

In related experiments, a “recombinantly-matured” VEGF-C polypeptide(VEGF-C ΔNΔCHis, described below) was shown to stimulate theincorporation of ³H-thymidine into the DNA of BCE cells in a dosedependent manner (VEGF-C concentrations of 0, 10, 100, and 1000 pMtested). This data tends to confirm the observation, under lightmicroscopy, that VEGF-C stimulates proliferation of these cells.

Example 16 VEGF-C is Expressed in Multiple Tissues

Northern blots containing 2 micrograms of isolated poly(A)⁺RNA frommultiple human tissues (blot from Clontech Laboratories, Inc., PaloAlto, Calif.) were probed with radioactively labeled insert of the 2.1kb VEGF-C cDNA clone. Northern blotting and hybridization analysisshowed that the 2.4 kb RNA and smaller amounts of a 2.0 kb mRNA areexpressed in multiple human tissues, most prominently in the heart,placenta, muscle, ovary and small intestine, and less prominently inprostate, colon, lung, pancreas, and spleen. Very little VEGF-C RNA wasseen in the brain, liver, kidney, testis, or thymus and peripheral bloodleukocytes (PBL) appeared negative. A similar analysis of RNA from humanfetal brain, lung, liver, and kidney tissues showed that VEGF-C ishighly expressed in the kidney and lung and to a lesser degree in theliver, while essentially no expression is detected in the brain.Interestingly, VEGF expression correlates with VEGF-C expression inthese tissues, whereas VEGF-B is highly expressed in all four fetaltissues analyzed.

Example 17 The VEGF-C Gene Localizes to Chromosome 4q34

A DNA panel of 24 interspecies somatic cell hybrids, which had retainedone or two human chromosomes, was used for the chromosomal localizationof the VEGF-C gene (Bios Laboratories, Inc., New Haven, Conn.). DNAsfrom human rodent somatic cell hybrids containing defined sets of humanchromosomes were analyzed by Southern blotting and hybridization with aVEGF-C cDNA probe. Among 24 DNA samples on the hybrid panel,representing different human chromosomes, human-specific signals wereobserved only in hybrids which contained human chromosome 4. The resultswere confirmed by PCR of somatic cell hybrid DNAs using VEGF-C specificprimers, where amplified bands were obtained only from DNAs containinghuman chromosome 4.

A genomic P1 plasmid for VEGF-C was isolated using specific primers andPCR and verified by Southern blotting and hybridization using a VEGF-Cspecific cDNA probe. The chromosomal localization of VEGF-C was furtherstudied using metaphase FISH. Using the P1 probe for VEGF-C in FISH, aspecific hybridization to the 4q34 chromosomal band was detected in 40out of 44 metaphases. Double-fluorochrome hybridization using a cosmidprobe specific for the aspartylglucosaminidase (AGA) gene showed thatVEGF-C is located just proximal to the AGA gene previously mapped to the4q34-35 chromosomal band.

Biotin-labeled VEGF-C P1 and digoxigenin-labeled AGA cosmid probes werehybridized simultaneously to metaphase chromosomes. This experimentdemonstrated that the AGA gene is more telomerically located than theVEGF-C gene. The foregoing example demonstrates the utility ofpolynucleotides of the invention as chromosomal markers and for thepresence or absence of the VEGF-C gene region in normal or diseasedcells. The VEGF-C locus at 4q34 is a candidate target for mutationsleading to vascular malformations or cardiovascular diseases.

Example 18 Effect of Glucose Concentration and Hypoxia on VEGF, VEGF-Band VEGF-C mRNA Levels in C6 Glioblastoma Cells

Confluent cultures of C6 cells (ATCC CCL 107) were grown on 10 cmdiameter tissue culture plates containing 2.5 ml of DMEM and 5% fetalcalf serum plus antibiotics. The cultures were exposed for 16 hours tonormoxia in a normal cell culture incubator containing 5% CO₂ or hypoxiaby closing the culture plates in an airtight glass chamber and burning apiece of wood inside until the flame was extinguished due to lack ofoxygen. Polyadenylated RNA was isolated (as in the other examples), and8 micrograms of the RNA was electrophoresed and blot-hybridized with amixture of the is VEGF, VEGF-B and VEGF-C probes. The results show thathypoxia strongly induces VEGF mRNA expression, both in low and highglucose, but has no significant effect on the VEGF-B mRNA levels. TheVEGF-C mRNA isolated from hypoxic cells runs slightly faster in gelelectrophoresis and an extra band of faster mobility can be seen belowthe upper mRNA band. This observation suggests that hypoxia affectsVEGF-C RNA processing. One explanation for this observation is thatVEGF-C mRNA splicing is altered, affecting the VEGF-C open reading frameand resulting in an alternative VEGF-C protein being produced by hypoxiccells. Such alternative forms of VEGF-C and VEGF-C-encodingpolynucleotides are contemplated as an aspect of the invention. Thisdata indicates screening and diagnostic utilities for polynucleotidesand polypeptides of the invention, such as methods whereby a biologicalsample is screened for the hypoxia-induced form of VEGF-C and/or VEGF-CmRNA. The data further suggests a therapeutic indication for antibodiesand/or other inhibitors of the hypoxia-induced form of VEGF-C or thenormal form of VEGF-C.

Example 19 Pulse-Chase Labeling and Immunoprecipitation of VEGF-CPolypeptides from 293 EBNA Cells Transfected with VEGF-C ExpressionVector

The following VEGF-C branched amino-terminal peptide, designated PAM126, was synthesized for production of anti-VEGF-C antiserum:

NH₂-E-E-T-I-K-F-A-A-A-H-Y-N-T-E-I-L-K-COOH (SEQ ID NO: 9). Inparticular, PAM126 was synthesized as a branched polylysine structureK3PA4 having four peptide acid (PA) chains attached to two availablelysine (K) residues. The synthesis was performed on a 433A PeptideSynthesizer (Applied Biosystems) using Fmoc-chemistry and TentaGel S MAPRAM10 resin mix (RAPP Polymere GmbH, Tubingen, Germany), yielding bothcleavable and resin-bound peptides. The cleavable peptide was purifiedvia reverse phase HPLC and was used together with the resin-boundpeptide in immunizations. The correctness of the synthesis products wereconfirmed using mass-spectroscopy (Lasermatt).

The PAM126 peptide was dissolved in phosphate buffered saline (PBS),mixed with Freund's adjuvant, and used for immunization of rabbits atbi-weekly intervals using methods standard in the art (Harlow and Lane,Antibodies, a laboratory manual, Cold Spring Harbor Laboratory Press(1988)). Antisera obtained after the fourth booster immunization wasused for immunoprecipitation of VEGF-C in pulse-chase experiments, asdescribed below.

For pulse-chase analysis, 293 EBNA cells transfected with a VEGF-Cexpression vector (i.e., the FLT4-L cDNA inserted into the pREP7expression vector as described above) were incubated for 30 minutes inmethionine-free, cysteine-free, serum-free DMEM culture medium at 37° C.The medium was then changed, and 200 μCi of Pro-mix™ (Amersham), wasadded. The cell layers were incubated in this labeling medium for twohours, washed with PBS, and incubated for 0, 15, 30, 60, 90, 120, or 180minutes in serum-free DMEM (chase). After the various chase periods, themedium was collected, the cells were again washed two times in PBS, andlysed in immunoprecipitation buffer. The VEGF-C polypeptides wereanalyzed from both the culture medium and from the cell lysates byimmunoprecipitation, using the VEGF-C-specific antiserum raised againstthe NH₂-terminal peptide (PAM126) of the 23 kD VEGF-C form.Immunoprecipitated polypeptides were analyzed via SDS-PAGE followed byautoradiography.

The resultant autoradiograms demonstrated that immediately after a 2hour labeling (chase time 0), the VEGF-C vector-transfected cellscontained a radioactive polypeptide band of about 58 kD (originallyestimated to be about 55 kD, and re-evaluated to be about 58 kD usingdifferent size standards), which was not observed in mock-transfectedcells (M). Most of this ˜58 kD precursor undergoes dimerization. This˜58 kD polypeptide band gradually diminished in intensity withincreasing chase periods. A 32 kD polypeptide band also is observed inVEGF-C transfected cells (but not mock-transfected cells). This 32 kDband disappears from cells with similar kinetics to that of the ˜58 kDband. Additional analysis indicated that the 32 kD band was a doublet of29 kD and 31-32 kD forms, held together by disulfide bonds.Simultaneously, increasing amounts of 32 kD and subsequently 23 kD and14-15 kD polypeptides appeared in the medium.

Collectively, the data from the pulse-chase experiments indicate thatthe ˜58 kD intracellular polypeptide represents a pro-VEGF-Cpolypeptide, which is proteolytically cleaved either intracellularly orat the cell surface into the 29 kD and 31-32 kD polypeptides. The 29/31kD form is secreted and simultaneously further processed by proteolysisinto the 23 kD and 14-15 kD forms. In additional experiments, disulfidelinked dimers of the 29 kD and 15 kD forms were observed. Withoutintending to be limited to a particular theory, it is believed thatprocessing of the VEGF-C precursor occurs as removal of a signalsequence, removal of the COOH-terminal domain (BR3P), and removal of anamino terminal polypeptide, resulting in a VEGF-C polypeptide having theTEE . . . amino terminus.

At high resolution, the 23 kD polypeptide band appears as aclosely-spaced polypeptide doublet, suggesting heterogeneity in cleavageor glycosylation.

Example 20 Isolation of Mouse and Quail cDNA Clones Encoding VEGF-C

A. Murine VEGF-C

To clone a murine VEGF-C, approximately 1×10⁶ bacteriophage lambdaclones of a commercially-available 12 day mouse embryonal cDNA library(lambda EXlox library, Novagen, catalog number 69632-1) were screenedwith a radiolabeled fragment of human VEGF-C cDNA containing nucleotides495 to 1661 of SEQ ID NO: 7. One positive clone was isolated.

A 1323 by EcoRI HindIII fragment of the insert of the isolated mousecDNA clone was subcloned into the corresponding sites of the pBluescriptSK+ vector (Stratagene) and sequenced. The cDNA sequence of this clonewas homologous to the human VEGF-C sequence reported herein, except thatabout 710 by of 5′-end sequence present in the human clone was notpresent in the mouse clone.

For further screening of mouse cDNA libraries, a HindIII-BstXI (HindIIIsite is from the pBluescript SK+ polylinker) fragment of 881 by from thecoding region of the mouse cDNA clone was radiolabeled and used as aprobe to screen two additional mouse cDNA libraries. Two additional cDNAclones from an adult mouse heart ZAP II cDNA library (Stratagene,catalog number 936306) were identified. Three additional clones alsowere isolated from a mouse heart 5′-stretch-plus cDNA library in λgt11(Clontech Laboratories, Inc., catalog number ML5002b). Of the latterthree clones, one was found to contain an insert of about 1.9 kb. Theinsert of this cDNA clone was subcloned into EcoRI sites of pBluescriptSK+ vector and both strands of this clone were completely sequenced,resulting in the nucleotide and deduced amino acid sequences shown inSEQ ID NOs: 10 and 11.

It is contemplated that the polypeptide corresponding to SEQ ID NO: 11is processed into a mature mouse VEGF-C protein, in a manner analogousto the processing of the human VEGF-C prepropeptide. Putative cleavagesites for the mouse protein are identified using procedures outlinedabove for identification of cleavage sites for the human VEGF-Cpolypeptide.

The foregoing results demonstrate the utility of polynucleotides of theinvention for identifying and isolating polynucleotides encoding othernon-human mammalian VEGF-C proteins. Such identified and isolatedpolynucleotides, in turn, can be expressed (using procedures similar tothose described in preceding examples) to produce recombinantpolypeptides corresponding to non-human mammalian forms of VEGF-C.

B. Quail VEGF-C

The mouse and human VEGF-C sequences were used to design probes forisolating a quail VEGF-C cDNA from a quail cDNA library. A fragment ofthe human VEGF-C cDNA comprising nucleotides 495-1670 of SEQ ID NO 7 wasobtained by PCR amplification, cloned into the pCR11 vector (Invitrogen)according to the manufacturer's instructions, and amplified. The insertwas isolated by EcoRI digestion and preparative gel electrophoresis andthen labeled using radioactive dCTP and random priming. A cDNA librarymade from quail embryos of stage E-4 in pcDNA-1 vector (Invitrogen) wasthen screened using this probe. About 200,000 colonies were plated andfilter replicas were hybridized with the radioactive probe. Ninepositive clones were identified and secondarily plated. Two of the nineclones hybridized in secondary screening. The purified clones (clones 1and 14) had approximately 2.7 kb EcoRI inserts. Both clones wereamplified and then sequenced using the T7 and SP6 primers (annealing tothe vector). In addition, an internal SphI restriction endonucleasecleavage site was identified about 1.9 kb from the T7 primer side of thevector and used for subcloning 5′- and 3′-SphI fragments, followed bysequencing from the SphI end of the subclones. The sequences obtainedwere identical from both clones and showed a high degree of similarityto the human VEGF-C coding region. Subsequently, walking primers weremade in both directions and double-stranded sequencing was completed for1743 base pairs, including the full-length open reading frame.

The cDNA sequence obtained includes a long open reading frame and 5′untranslated region. The DNA and deduced amino acid sequences for thequail cDNA are set forth in SEQ ID NOs: 12 and 13, respectively. Studiesperformed with the putative quail VEGF-C cDNA have shown that itsprotein product is secreted from transfected cells and interacts withavian VEGFR-3 and VEGFR-2, further confirming the conclusion that thecDNA encodes a quail VEGF-C protein. The proteins secreted from 293-EBNAcells transfected with quail VEGF-C cDNA were analyzed inimmunoprecipitation studies using the VEGF-C-specific polyclonalantisera generated against the PAM126 polypeptide (Example 19). Adoublet band of about 30-32 kD, and a band of about 22-23 kD, wereimmunoprecipitated from the transfected cells but not from controlcells. These immunoprecipitation studies thus provide a furtherindication that VEGF-C from non-human species is processed (from aprepro-VEGF-C form) in a manner analogous to the processing of humanVEGF-C. As shown in FIG. 5, the human, murine, and avian (quail) VEGF-Cprecursor amino acid sequences share a significant degree ofconservation. This high degree of homology between species permits theisolation of VEGF-C encoding sequences from other species, especiallyvertebrate species, and more particularly mammalian and avian species,using polynucleotides of the present invention as probes and usingstandard molecular biological techniques such as those described herein.

Example 21 N-Terminal Peptide Sequence Analyses of Recombinant VEGF-C

Cells (293 EBNA) transfected with VEGF-C cDNA (see Example 13) secreteseveral forms of recombinant VEGF-C (FIG. 6A, lane IP). In the absenceof alkylation, the three major, proteolytically-processed forms ofVEGF-C migrate in SDS-PAGE as proteins with apparent molecular masses of32/29 kD (doublet), 21 kD and 15 kD. Two minor polypeptides exhibitapproximate molecular masses of 63 and 52 kD, respectively. One of thesepolypeptides is presumably a glycosylated and non-processed form; theother polypeptide is presumably glycosylated and partially processed.More precise size measurements (using SDS-PAGE under reducingconditions) revealed that the molecular masses of the VEGF-C forms thatwere initially estimated as 63, 52, 32, 23, and 14 kD (using SDS-PAGEunder reducing conditions and a different set of size standards) areapproximately 58, 43, 31, 29, 21, and 15 kD, respectfully (the initialmeasurements in most cases falling within acceptable 10% error of themore precise measurements).

To determine sites of proteolytic cleavage of the VEGF-C precursor, animmunoaffinity column was used to purify VEGF-C polypeptides from theconditioned medium of 293 EBNA cells transfected with VEGF-C. cDNA. Toprepare the immunoaffinity column, a rabbit was immunized with asynthetic peptide corresponding to amino acids 104-120 of SEQ ID. NO: 8:H₂N-EETIKFAAAHYNTEILK (see PAM126 in Example 19). The IgG fraction wasisolated from the serum of the immunized rabbit using protein ASepharose (Pharmacia). The isolated IgG fraction was covalently bound toCNBr-activated Sepharose CL-4B (Pharmacia) using standard techniques ata concentration of 5 mg IgG/ml of Sepharose. This immunoaffinity matrixwas used to isolate processed VEGF-C from 1.2 liters of the conditionedmedium (CM).

The purified material eluted from the column was analyzed by gelelectrophoresis and Western blotting. Fractions containing VEGF-Cpolypeptides were combined, dialyzed against 10 mM Tris HCl,vacuum-dried, electrotransferred to Immobilon-P (polyvinylidenedifluoride or PVDF) transfer membrane (Millipore, Marlborough, Mass.)and subjected to N-terminal amino acid sequence analysis.

The polypeptide band of 32 kD yielded two distinct sequences:NH₂-FESGLDLSDA . . . and NH₂-AVVMTQTPAS . . . (SEQ ID NO: 14), theformer corresponding to the N-terminal part of VEGF-C after cleavage ofthe signal peptide, starting from amino acid 32 (SEQ ID NO: 8), and thelatter corresponding to the kappa-chain of IgG, which was present in thepurified material due to “leakage” of the affinity matrix during theelution procedure.

In order to obtain the N-terminal peptide sequence of the 29 kD form ofVEGF-C, a construct (VEGF-C NHis) encoding a VEGF-C mutant wasgenerated. In particular, the construct encoded a VEGF-C mutant thatfused a 6×His tag to the N-terminus of the secreted precursor (i.e.,between amino acids 31 and 33 in SEQ ID NO: 8). The phenylalanine atposition 32 was removed to prevent possible cleavage of the tag sequenceduring secretion of VEGF-C. The VEGF-C NHis construct was cloned intopREP7 as a vector; the construction is described more fully in Example28, below.

The calcium phosphate co-precipitation technique was used to transfectVEGF-C NHis into 293 EBNA cells. Cells were incubated in DMEM/10% fetalcalf serum in 15 cm cell culture dishes (a total of 25 plates). Thefollowing day, the cells were reseeded into fresh culture dishes (75plates) containing the same medium and incubated for 48 hours. Celllayers were then washed once with PBS and DMEM medium lacking. FCS wasadded. Cells were incubated in this medium for 48 hours and the mediumwas collected, cleared by centrifugation at 5000×g and concentrated 500×using an Ultrasette Tangential Flow Device (Filtron, Northborough,Mass.), as described in Example 5 above. VEGF-C NHis was purified fromthe concentrated conditioned medium using TALON™ Metal Affinity Resin(Clontech Laboratories, Inc.) and the manufacturer's protocol for nativeprotein purification using imidazole-containing buffers. The protein waseluted with a solution containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl,and 200 mM imidazole. The eluted fractions containing purified VEGF-CNHis were detected by immunoblotting with Antiserum 882 (antiserum fromrabbit 882, immunized with the PAM-126 polypeptide). Fractionscontaining VEGF-C NHis were combined, dialyzed and vacuum-dried. Due toto the presence of the 6×His tag at the N-terminus of this form ofVEGF-C, the upper component of the major doublet of the VEGF-C NHismigrates slightly slower than the 32 kD form of wild type VEGF-C,thereby improving the separation of the VEGF-C NHis 32 kD mutant fromthe 29 kD band using SDS-PAGE. Approximately 15 μg of the purifiedVEGF-C were subjected to SDS-PAGE under reducing conditions,electrotransferred to Immobilon-P(PVDF) transfer membrane (Millipore,Inc., Marlborough, Mass.) and the band at 29 kD was subjected toN-terminal amino acid sequence analysis. This sequence analysis revealedan N-terminal sequence of H₂N-SLPAT . . . , corresponding to amino acids228-232 of VEGF-C (SEQ ID NO: 8).

The polypeptide band of 21 kD yielded the sequence H₂N-AHYNTEILKS . . ., corresponding to an amino-terminus starting at amino acid 112 of SEQID NO: 8. Thus, the proteolytic processing site which results in the 21kD form of VEGF-C produced by transfected 293 EBNA cells apparentlyoccurs nine amino acid residues downstream of the cleavage site whichresults in the 23 kD form of VEGF-C secreted by PC-3 cells.

The N-terminus of the 15 kD form was identical to the N-terminus of the32 kD form (NH₂-FESGLDLSDA . . . ). The 15 kD form was not detected whenrecombinant VEGF-C was produced by COS cells. This suggests thatproduction of this form is cell lineage specific.

Example 22 Dimeric and Monomeric Forms of VEGF-C

The composition of VEGF-C dimers was analyzed as follows. Cells (293EBNA cells), transfected with the pREP7 VEGF-C vector as described inExample 11, were metabolically labeled with Pro-mix L-[³⁵S] labeling mix(Amersham Corp.) to a final concentration of 100

In parallel, a VEGF-C mutant, designated “R102S”, was prepared andanalyzed. To prepare the DNA encoding VEGF-C-R102S, the arginine codonat position 102 of SEQ ID NO: 8 was replaced with a serine codon. ThisVEGF-C-R102S-encoding DNA, in a pREP7 vector, was transfected into 293EBNA cells and expressed as described above. VEGF-C polypeptides wereimmunoprecipitated using antisera 882 (obtained by immunization of arabbit with a polypeptide corresponding to residues 104-120 of SEQ IDNO: 8 (see previous Example)) and antisera 905 (obtained by immunizationof a rabbit with a polypeptide corresponding to a portion of thepro-VEGF-C leader: H₂N-ESGLDLSDAEPDAGEATAYASK (residues 33 to 54 of SEQID NO: 8).

The immunoprecipitates from each cell culture were subjected to SDS-PAGEunder non-denaturing conditions (FIG. 6B). Bands 1-6 were cut out fromthe gel, soaked for 30 minutes in 1× gel-loading buffer containing 200mM β-mercaptoethanol, and individually subjected to SDS-PAGE underdenaturing conditions (FIGS. 6A and 6C, lanes 1-6).

As can be seen from FIGS. 6A-C, each high molecular weight form ofVEGF-C (FIG. 6B, bands 1-4) consists of at least two monomers bound bydisulfide bonds (Compare FIGS. 6A and 6C, lanes 1-4, in the reducinggels). The main component of bands 1-3 is the doublet of 32/29 kD, whereboth proteins are present in an equimolar ratio. The main fraction ofthe 21 kD form is secreted as either a monomer or as a homodimerconnected by means other than disulfide bonds (bands 6 and lanes 6 inFIGS. 6A-C).

The R102S mutation creates an additional site for N-linked glycosylationin VEGF-C at the asparagine residue at position 100 in SEQ ID NO: 8.Glycosylation at this additional glycosylation site increases theapparent molecular weight of polypeptides containing the site, asconfirmed in FIGS. 6A-C and FIGS. 7A-B. The additional glycosylationlowers the mobility of forms of VEGF-C-R102S that contain the additionalglycosylation site, when compared to polypeptides of similar primarystructure corresponding to VEGF-C. FIGS. 6A-C and FIGS. 7A-B reveal thatthe VEGF-C-R102S polypeptides corresponding to the 32 kD and 15 kD formsof wt VEGF-C exhibit increased apparent molecular weights, indicatingthat each of these polypeptides contains the newly introducedglycosylation site. In particular, the VEGF-C-R102S polypeptidecorresponding to the 15 kD polypeptide from VEGF-C comigrates on a gelwith the 21 kD form of the wild type (wt) VEGF-C, reflecting a shift onthe gel to a position corresponding to a greater apparent molecularweight. (Compare lanes 4 in FIGS. 6A and 6C). The mobility of the 58 kDform of VEGF-C was slowed to 64 kD by the R102S mutation, indicatingthat this form contains the appropriate N-terminal peptide of VEGF-C.The mobilities of the 21, 29, and 43 kD forms were unaffected by theR102S mutation, suggesting that these polypeptides contain peptidesequences located C-terminally of R₁₀₂.

In a related experiment, another VEGF-C mutant, designated “R226,227S,”was prepared and analyzed. To prepare a DNA encoding VEGF-C-R226,227S,the arginine codons at positions 226 and 227 of SEQ ID NO: 8 werereplaced with serine codons by site-directed mutagenesis. The resultantDNA was transfected into 293 EBNA cells as described above and expressedand analyzed under the same conditions as described for VEGF-C andVEGF-C-R102S. In the conditioned medium from the cells expressingVEGF-C-R226,227S, no 32 kD form of VEGF-C was detected. These resultsindicate that a C-terminal cleavage site of wild-type VEGF-C is adjacentto residues 226 and 227 of SEQ ID NO: 8, and is destroyed by themutation of the arginines to serines. Again, the mobility of the 29 kDcomponent of the doublet was unchanged (FIGS. 7A-B).

Taken together, these data indicate that the major form of the processedVEGF-C is a heterodimer consisting of (1) a polypeptide of 32 kDcontaining amino acids 32-227 of the prepro-VEGF-C (amino acids 32 to227 in SEQ ID NO: 8) attached by disulfide bonds to (2) a polypeptide of29 kD beginning with amino acid 228 in SEQ ID NO: 8. These data are alsosupported by a comparison of the pattern of immunoprecipitated, labeledVEGF-C forms using antisera 882 and antisera 905.

When VEGF-C immunoprecipitation was carried out using conditionedmedium, both antisera (882 and 905) recognized some or all of the threemajor processed forms of VEGF-C (32/29 kD, 21 kD and 15 kD). When theconditioned medium was reduced by incubation in the presence of 10 mMdithiothreitol for two hours at room temperature with subsequentalkylation by additional incubation with 25 mM iodoacetamide for 20minutes at room temperature, neither antibody precipitated the 29 kDcomponent, although antibody 882 still recognized polypeptides of 32 kD,21 kD and 15 kD. In subsequent experiments it was observed that neitherantibody was capable of immunoprecipitating the 43 kD form. Theseresults are consistent with the nature of the oligopeptide antigen usedto elicit the antibodies contained in antisera 882, an oligopeptidecontaining amino acid residues 104-120 of SEQ ID NO: 8. On the otherhand, antisera 905 recognized only the 32 kD and 15 kD polypeptides,which include sequence of the oligopeptide (amino acids 33 to 54 of SEQID NO: 8) used for immunization to obtain antisera 905. Taking intoaccount the mobility shift of the 32 kD and 15 kD forms, theimmunoprecipitation results with the R102S mutant were similar (FIGS.8A-B). The specificity of antibody 905 is confirmed by the fact that itdid not recognize a VEGF-C ΔN form wherein the N-terminal propeptidespanning residues 32-102 of the unprocessed polypeptide had been deleted(FIG. 8B).

The results of these experiments also demonstrate that the 21 kDpolypeptide is found (1) in heterodimers with other molecular forms (seeFIGS. 6A-C and FIGS. 7A-B), and (2) secreted as a monomer or a homodimerheld by bonds other than disulfide bonds (FIGS. 6A and 6B, lanes 6).

The experiments disclosed in this example demonstrate that several formsof VEGF-C exist. A variety of VEGF-C monomers were observed and thesemonomers can vary depending on the level and pattern of glycosylation.In addition, VEGF-C was observed as a multimer, for example a homodimeror a heterodimer. The processing of VEGF-C is schematically presented inFIG. 9 (disulfide bonds not shown). All forms of VEGF-C are within thescope of the present invention.

Example 23 In situ Hybridization of Mouse Embryos

To analyze VEGF-C mRNA distribution in different cells and tissues,sections of 12.5 and 14.5-day post-coitus (p.c.) mouse embryos wereprepared and analyzed via in situ hybridization using labeled VEGF-Cprobes. In situ hybridization of tissue sections was performed asdescribed in Västrik et al., J. Cell Biol., 128:1197-1208 (1995). Amouse VEGF-C antisense RNA probe was generated from linearizedpBluescript II SK+ plasmid (Stratagene Inc., La Jolla, Calif.),containing a cDNA fragment corresponding to nucleotides 499-979 of amouse VEGF-C cDNA (SEQ ID NO: 10). Radiolabeled RNA was synthesizedusing T7 polymerase and [³⁵S]-UTP (Amersham). Mouse VEGF-B antisense andsense RNA probes were synthesized in a similar manner from linearizedpCRII plasmid containing the mouse VEGF-B cDNA insert as describedOlofsson et al., Proc. Natl. Acad. Sci. (USA), 93:2576-2581 (1996). Thehigh stringency wash was for 45 minutes at 65° C. in a solutioncontaining 30 mM dithiothreitol (DTT) and 4×SSC. The slides were exposedfor 28 days, developed and stained with hematoxylin. For comparison,similar sections were hybridized with a VEGFR-3 probe and the 12.5-dayp.c. embryos were also probed for VEGF-B mRNA.

Darkfield and lightfield photomicrographs from these experiments arepresented in commonly-owned PCT patent application PCT/FI96/00427, filedAug. 1, 1996, published as WO 97/05250, incorporated by referenceherein. Observations from the photomicrographs are summarized below. Ina 12.5 day p.c. embryo, a parasagittal section revealed that VEGF-C mRNAwas particularly prominent in the mesenchyme around the vesselssurrounding the developing metanephros. In addition, hybridizationsignals were observed between the developing vertebrae, in thedeveloping lung mesenchyme, in the neck region and developing forehead.The specificity of these signals was evident from the comparison withVEGF-B expression in an adjacent section, where the myocardium gave avery strong signal and lower levels of VEGF-B mRNA were detected inseveral other tissues. Both genes appear to be expressed in between thedeveloping vertebrae, in the developing lung, and forehead.Hybridization of the VEGF-C sense probe showed no specific expressionwithin these structures.

Studies also were conducted of the expression patterns of VEGF-C andVEGFR-3 in 12.5 day p.c. mouse embryos in the jugular region, where thedeveloping to dorsal aorta and cardinal vein are located. This is thearea where the first lymphatic vessels sprout from venous sac-likestructures according to the long-standing theory of Sabin, Am. J. Anat.,9:43-91 (1909). An intense VEGF-C signal was detected in the mesenchymesurrounding the developing venous sacs which also were positive forVEGFR-3.

The mesenterium supplies the developing gut with blood and containsdeveloping lymphatic vessels. The developing 14.5 day p.c. mesenteriumis positive for VEGF-C mRNA, with particularly high expression inconnective tissue surrounding certain vessels. The adjacent mesenterialVEGFR-3 signals that were observed originate from small capillaries ofthe mesenterium. Therefore, there appears to be a paracrine relationshipbetween the production of the mRNAs for VEGF-C and its receptor. Thisdata indicates that VEGF-C is expressed in a variety of tissues.Moreover, the pattern of expression is consistent with a role for VEGF-Cin venous and lymphatic vessel development. Further, the data revealsthat VEGF-C is expressed in non-human animals.

Example 24 Analysis of VEGF, VEGF-B, and VEGF-C mRNA Expression in Fetaland Adult Tissues

A human fetal tissue Northern blot containing 2 mg of polyadenylatedRNAs from brain, lung, liver and kidney (Clontech Inc.) was hybridizedwith a pool of the following probes: a human full-length VEGF-C cDNAinsert (Genbank Acc. No. X94216), a human VEGF-B₁₆₇ cDNA fragment(nucleotides 1-382, Genbank Acc. No. U48800) obtained by PCRamplification; and a human VEGF 581 by cDNA fragment covering base pairs57-638 (Genbank Acc. No. X15997). Blots were washed under stringentconditions, using techniques standard in the art.

Mouse embryo multiple tissue Northern blot (Clontech Inc.) containing 2μg of polyadenylated RNAs from 7, 11, 15 and 17 day postcoital (p.c.)embryos was hybridized with mouse VEGF-C cDNA fragment (base pairs499-656). A mouse adult tissue Northern blot was hybridized with theprobes for human VEGF, VEGF-B₁₆₇, VEGF-C and with a VEGFR-3 cDNAfragment (nucleotides 1-595; Genbank Acc. No X68203).

In adult mouse tissues, both 2.4 kb and 2.0 kb mRNA signals wereobserved with the VEGF-C probe, at an approximately 4:1 ratio. The mostconspicuous signals were obtained from lung and heart RNA, while kidney,liver, brain, and skeletal muscle had lower levels, and spleen andtestis had barely visible levels. As in the human tissues, VEGF mRNAexpression in adult mice was most abundant in lung and heart RNA,whereas the other samples showed less coordinate regulation with VEGF-Cexpression. Skeletal muscle and heart tissues gave the highest VEGF-BmRNA levels from adult mice, as previously reported Olofsson et al.,Proc. Natl. Acad. Sci. (USA), 93:2576-2581 (1996). Comparison withVEGFR-3 expression showed that the tissues where VEGF-C is expressedalso contain mRNA for its cognate receptor tyrosine kinase, although inthe adult liver VEGFR-3 mRNA was disproportionally abundant.

To provide a better insight into the regulation of the VEGF-C mRNAduring embryonic development, polyadenylated RNA isolated from mouseembryos of various gestational ages (7, 11, 15, and 17 day p.c.) washybridized with the mouse VEGF-C probe. These analyses showed that theamount of 2.4 kb VEGF-C mRNA is relatively constant throughout thegestational period.

Example 25 Regulation of mRNAs for VEGF Family Members by Serum,Interleukin-1 and Dexamethasone in Human Fibroblasts in Culture

Human IMR-90 fibroblasts were grown in DMEM medium containing 10% FCSand antibiotics. The cells were grown to 80% confluence, then starvedfor 48 hours in 0.5% FCS in DMEM. Thereafter, the growth medium waschanged to DMEM containing 5% FCS, with or without 10 ng/mlinterleukin-1 (IL-1) and with or without 1 mM dexamethasone. The cultureplates were incubated with these additions for the times indicated, andtotal cellular RNA was isolated using the TRIZOL kit (GIBCO-BRL). About20 μg of total RNA from each sample was electrophoresed in 1.5%formaldehyde-agarose gels as described in Sambrook et al., supra (1989).The gel was used for Northern blotting and hybridization withradiolabeled insert DNA from the human VEGF clone (a 581 by cDNAcovering bps 57-638, Genbank Acc. No. 15997) and a human VEGF-B₁₆₇ cDNAfragment (nucleotides 1-382, Genbank Acc. No. U48800). Subsequently, theNorthern blots were probed with radiolabeled insert from the VEGF-C cDNAplasmid. Primers were labeled using a standard technique involvingenzymatic extension reactions of random primers, as would be understoodby one of ordinary skill in the art.

The Northern blot analyses revealed that very low levels of VEGF-C andVEGF are expressed by the starved IMR-90 cells as well as cells after 1hour of stimulation. In contrast, abundant VEGF-B mRNA signal wasvisible under these conditions. After 4 hours of serum stimulation,there was a strong induction of VEGF-C and VEGF mRNAs, which werefurther increased in the IL-1 treated sample: The effect of IL-1 seemedto be abolished in the presence of dexamethasone. A similar pattern ofenhancement was observed in the 8 hour sample, but a gradualdown-regulation of all signals was observed for both RNAs in the 24 hourand 48 hour samples. In contrast, VEGF-B mRNA levels remained constantand thus showed remarkable stability throughout the time period. Theresults are useful in guiding efforts to use VEGF-C and its fragments,its antagonists, and anti-VEGF-C antibodies in methods for treating avariety of disorders.

Example 26 Expression and Analysis of Recombinant Murine VEGF-C

The mouse VEGF-C cDNA was expressed as a recombinant protein and thesecreted protein was analyzed for its receptor binding properties. Thebinding of mouse VEGF-C to the human VEGFR-3 extracellular domain wasstudied by using media from Bosc23 cells transfected with mouse VEGF-CcDNA in a retroviral expression vector.

The 1.8 kb mouse VEGF-C cDNA was cloned as an EcoRI fragment into theretroviral expression vector pBabe-puro containing the SV40 earlypromoter region [Morgenstern et al., Nucl. Acids Res., 18:3587-3595(1990)], and transfected into the Bosc23 packaging cell line [Pearet etal., Proc. Natl. Acad. Sci. (USA), 90:8392-8396 (1994)] by thecalcium-phosphate precipitation method. For comparison, Bosc23 cellsalso were transfected with the previously-described human VEGF-Cconstruct in the pREP7 expression vector. The transfected cells werecultured for 48 hours prior to metabolic labeling. Cells were changedinto DMEM medium devoid of cysteine and methionine, and, after 45minutes of preincubation and medium change, Pro-mix™ L-[³⁵S] in vitrocell labeling mix (Amersham Corp.), in the same medium, was added to afinal concentration of about 120 μCi/ml. After 6 hours of incubation,the culture medium was collected and clarified by centrifugation.

For immunoprecipitation, 1 ml aliquots of the media frommetabolically-labeled Bosc23 cells transfected with empty vector ormouse or human recombinant VEGF-C, respectively, were incubatedovernight on ice with 2 μl of rabbit polyclonal antiserum raised againstan N-terminal 17 amino acid oligopeptide of mature humanVEGF-C(H₂N-EETIKFAAAHYNTEMK) (SEQ ID NO: 8, residues 104-120).Thereafter, the samples were incubated with protein A sepharose for 40minutes at 4° C. with gentle agitation. The sepharose beads were thenwashed twice with immunoprecipitation buffer and four times with 20 mMTris-HCl, pH 7.4. Samples were boiled in Laemmli buffer and analyzed by12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).

Immunoprecipitation of VEGF-C from media of transfected andmetabolically-labeled cells revealed bands of approximately 30-32×10³M_(r) (a doublet) and 22-23×10³ M_(r) in 12.5% SDS-PAGE. These bandswere not detected in samples from nontransfected or mock-transfectedcells. These results show that antibodies raised against human VEGF-Crecognize the corresponding mouse ligand, and provide an indication thatthe proteolytic processing that occurs to produce murine VEGF-C isanalogous to the processing that occurs to produce human VEGF-C.

For receptor binding experiments, 1 ml aliquots of media frommetabolically-labeled Bosc23 cells were incubated with VEGFR-3extracellular domain (see Example 3), covalently coupled to sepharose,for 4 hours at 4° C. with gentle mixing. The sepharose beads were washedfour times with ice-cold phosphate buffered saline (PBS), and thesamples were analyzed by gel electrophoresis as described in Joukov etal., EMBO J, 15:290-298 (1996).

Similar 30-32×10³ M_(r) doublet and 22-23×10³M_(r) polypeptide bandswere obtained in the receptor binding assay as compared to theimmunoprecipitation assay. Thus, mouse VEGF-C binds to human VEGFR-3.The slightly faster mobility of the mouse VEGF-C polypeptides that wasobserved may be caused by the four amino acid residue differenceobserved in sequence analysis (residues H88-E91, FIG. 10).

The capacity of mouse recombinant VEGF-C to induce VEGFR-3autophosphorylation was also investigated. For the VEGFR-3 receptorstimulation experiments, subconfluent NIH 3T3-Flt4 cells, Pajusola etal., Oncogene, 9:3545-3555 (1994), were starved overnight in serum-freemedium containing 0.2% BSA. In general, the cells were stimulated withthe conditioned medium from VEGF-C vector-transfected cells for 5minutes, washed three times with cold PBS containing 200 μM vanadate,and lysed in RIPA buffer for immunoprecipitation analysis. The lysateswere centrifuged for 25 minutes at 16000×g and the resultingsupernatants were incubated for 2 hours on ice with the specificantisera, followed by immunoprecipitation using protein A-sepharose andanalysis in 7% SDS-PAGE. Polypeptides were transferred to nitrocelluloseand analyzed by immunoblotting using anti-phosphotyrosine (TransductionLaboratories) and anti-receptor antibodies, as described by Pajusola etal., Oncogene, 9:3545-3555 (1994). Filter stripping was carried out at50° C. for 30 minutes in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mMTris-HCl, pH 6.7, with occasional agitation. The results of theexperiment demonstrated that culture medium containing mouse VEGF-Cstimulates the autophosphorylation of VEGFR-3 to a similar extent ashuman baculoviral VEGF-C or the tyrosyl phosphatase inhibitorpervanadate.

Mouse VEGF-C appeared to be a potent inducer of VEGFR-3autophosphorylation, with the 195×10³ M_(r) precursor andproteolytically-cleaved 125×10³ M_(r) tyrosine kinase polypeptides ofthe receptor (Pajusola et al., Oncogene, 9:3545-3555 (1994)), beingphosphorylated.

VEGFR-2 stimulation was studied in subconfluent porcine aorticendothelial (PAE) cells expressing KCDR (VEGFR-2)(PAE-VEGFR-2)[Waltenberger et al., J. Biol. Chem., 269:26988-26995 (1994)], whichwere starved overnight in serum-free medium containing 0.2% BSA.Stimulation was carried out and the lysates prepared as described above.For receptor immunoprecipitation, specific antiserum for VEGFR-2[Waltenberger et al., J. Biol. Chem., 269:26988-26995 (1994)] was used.The inimunoprecipitates were analyzed as described for VEGFR-3 in 7%SDS-PAGE followed by Western blotting with anti-phosphotyrosineantibodies, stripping of the filter, and re-probing it with anti-VEGFR-2antibodies (Santa Cruz). VEGFR-2 stimulation was first tried withunconcentrated medium from cells expressing recombinant VEGF-C, butimmunoblotting analysis did not reveal any receptor autophosphorylation.

To further determine whether mouse recombinant VEGF-C can also induceVEGFR-2 autophosphorylation as observed for human VEGF-C, PAE cellsexpressing VEGFR-2 were stimulated with tenfold concentrated medium fromcultures transfected with mouse VEGF-C expression vector andautophosphorylation was analyzed. For comparison, cells treated withtenfold concentrated medium containing human recombinant VEGF-C (Joukovet al., (1996)), unconcentrated medium from human VEGF-C baculovirusinfected insect cells, or pervanadate (a tyrosyl phosphatase inhibitor)were used. In response to human baculoviral VEGF-C as well aspervanadate treatment, VEGFR-2 was prominently phosphorylated, whereashuman and mouse recombinant VEGF-C gave a weak and barely detectableenhancement of autophosphorylation, respectively. Media from cellcultures transfected with empty vector or VEGF-C cloned in the antisenseorientation did not induce autophosphorylation of VEGFR-2. Therefore,mouse VEGF-C binds to VEGFR-3 and activates this receptor at a muchlower concentration than needed for the activation of VEGFR-2.Nevertheless, the invention comprehends methods for using the materialsof the invention to take advantage of the interaction of VEGF-C withVEGFR-2, in addition to the interaction between VEGF-C and VEGFR-3.

Example 27 VEGF-C E104-S213 Fragment Expressed in Pichia YeastStimulates Autophosphorylation of Flt4 (VEGFR-3) and KDR (VEGFR-2)

A truncated form of human VEGF-C cDNA was constructed wherein (1.) thesequence encoding residues of a putative mature VEGF-C amino terminusH₂N-E(104)ETIK (SEQ ID NO: 8, residues 104 et seq.) was fused in-frameto the yeast PHO1 signal sequence (Invitrogen Pichia Expression Kit,Catalog #K1710-01), and (2) a stop codon was introduced after amino acid213 (H₂N— . . . RCMS; i.e., after codon 213 of SEQ ID NO: 7). Theresultant truncated cDNA construct was then inserted into the Pichiapastoris expression vector pHIL-S1 (Invitrogen). For the cloning, aninternal BglII site in the VEGF-C coding sequence was mutated withoutchange of the encoded polypeptide sequence.

This VEGF-C expression vector was then transfected into Pichia cells andpositive clones were identified by screening for the expression ofVEGF-C protein in the culture medium by Western blotting. One positiveclone was grown in a 50 ml culture, and induced with methanol forvarious periods of time from 0 to 60 hours. About 10 μl of medium wasanalyzed by gel electrophoresis, followed by Western blotting anddetection with anti-VEGF-C antiserum, as described above. Anapproximately 24 kD polypeptide (band spreading was observed due toglycosylation) accumulated in the culture medium of cells transfectedwith the recombinant VEGF-C construct, but not in the medium ofmock-transfected cells or cells transfected with the vector alone.

The medium containing the recombinant VEGF-C protein was concentrated byCentricon 30 kD cutoff ultrafiltration and used to stimulate NIH 3T3cells expressing Flt4 (VEGFR-3) and porcine aortic endothelial (PAE)cells expressing KDR (VEGFR-2). The stimulated cells were lysed andimmunoprecipitated using VEGFR-specific antisera and theimmunoprecipitates were analyzed by Western blotting usinganti-phosphotyrosine antibodies, chemiluminescence, and fluorography. Asa positive control for maximal autophosphorylation of the VEGFRs,vanadate (VO₄) treatment of the cells for 10 minutes was used. Mediumfrom Pichia cultures secreting the recombinant VEGF-C polypeptideinduced autophosphorylation of both Flt41 polypeptides of 195 kD and 125kD as well as the KDR polypeptide of about 200 kD. Vanadate, on theother hand, induces heavy tyrosyl phosphorylation of the receptor bandsin addition to other bands probably coprecipitating with the receptors.

These results demonstrate that a VEGF-homologous domain of VEGF-Cconsisting of amino acid residues 104E-213S (SEQ ID NO: 8, residues104-213) can be recombinantly produced in yeast and is capable ofstimulating the autophosphorylation of Flt4 (VEGFR-3) and KDR (VEGFR-2).Recombinant VEGF-C fragments such as the fragment described herein,which are capable of stimulating Flt4 or KDR autophosphorylation areintended as aspects of the invention; methods of using these fragmentsare also within the scope of the invention.

Example 28 Properties of the Differentially Processed Forms of VEGF-C

The following oligonucleotides were used to generate a set of VEGF-Cvariants and analogs:

5′-TCTCTTCTGTGCTTGAGTTGAG-3′ (SEQ ID NO: 15), used to generate VEGF-CR102S (arginine mutated to serine at position 102 (SEQ ID NO: 8));

5′-TCTCTTCTGTCCCTGAGTTGAG-3′ (SEQ ID NO: 16), used to generate VEGF-CR102G (arginine mutated to glycine at position 102 (SEQ ID NO: 8));

5′-TGTGCTGCAGCAAATTTTATAGTCTCTTCTGTGGCGGCGGC GGCGGCGGGCGCCTCGCGAGGACC-3′(SEQ ID NO: 17), used to generate VEGF-C ΔN (deletion of N-terminalpropeptide corresponding to amino acids 32-102 (SEQ ID NO: 8));

5′-CTGGCAGGGAACTGCTAATAATGGAATGAA-3′ (SEQ ID NO: 18), used to generateVEGF-C R226,227S (arginine codons mutated to serines at positions 226and 227 (SEQ ID NO: 8));

5′-GGGCTCCGCGTCCGAGAGGTCGAGTCCGGACTCGTGATGGTGATGGTGATGGGCGGCGGCGGCGGCGGGCGCCTCGCGAGGACC-3′ (SEQ ID NO: 19), used togenerate VEGF-C NHis (this construct encodes a polypeptide with a 6×Histag fused to approximately the N-terminus of the secreted precursor, asdescribed in Example 21 (amino acid 33 of SEQ ID NO: 8)).

Some of the foregoing VEGF-C mutant constructs were further modified toobtain additional constructs. For example, VEGF-C R102G in pALTER(Promega) and oligonucleotide 5′-GTATTATAATGTCCTCCACCAAATTTTATAG-3′ (SEQID NO: 20) were used to generate VEGF-C 4G, which encodes a polypeptidewith four point mutations: R102G, A110G, A111G, and A112G (alaninesmutated to glycines at positions 110-112 (SEQ ID NO: 8). These fourmutations are adjacent to predicted sites of cleavage of VEGF-Cexpressed in PC-3 and recombinantly expressed in 293 EBNA cells.

Another construct was created using VEGF-C ΔN and oligonucleotide5′-GTTCGCTGCCTGACACTGTGGTAGTGTTGCTGGCGGCCGCTAGTGATGGTGATGGTGATGAATAATGGAATGAACTTGTCTGTAAA CATCCAG-3′ (SEQ IDNO: 21) to generate VEGF-C ΔNΔCHis. This construct encodes a polypeptidewith a deleted N-terminal propeptide (amino acids 32-102); a deletedC-terminal propeptide (amino acids 226-419 of SEQ ID NO: 8); and anadded 6×His tag at the C-terminus (see SEQ ID NO: 59).

All constructs were further digested with HindIII and NotI, subclonedinto HindIII/NotI digested pREP7 vector, and used to transfect 293 EBNAcells. About 48 hours after transfection, the cells were eithermetabolically labelled with Pro-mix™ as described above, or starved inserum-free medium for 2 days. Media were then collected and used insubsequent experiments. Wild type (wt) VEGF-C, VEGF-C NHis and VEGF-CΔNΔCHis were expressed to similar levels in 293 EBNA cells. At the sametime, expression of the VEGF-C 4G polypeptide was considerably lower,possibly due to the changed conformation and decreased stability of thetranslated product. However, all the above VEGF-C mutants were secretedfrom the cells.

The conditioned media from the transfected and starved cells wereconcentrated 5-fold and used to assess their ability to stimulatetyrosine phosphorylation of Flt4 (VEGFR-3) expressed in NIH 3T3 cellsand KDR (VEGFR-2) expressed in PAE cells. Wild type (wt) VEGF-C, as wellas all three mutant polypeptides, stimulated 10 tyrosine phosphorylationof VEGFR-3. The most prominent stimulation observed was by the shortmature VEGF-C ΔNΔCHis. This mutant, as well as VEGF-C NHis, alsostimulated tyrosine phosphorylation of VEGFR-2. Thus, despite the factthat a major component of secreted recombinant VEGF-C is a dimer of32/29 kD, the active part of VEGF-C responsible for its binding toVEGFR-3 and VEGFR-2 is localized between amino acids 102 and 226 (SEQ IDNO: 8) of the VEGF-C precursor. Analysis and comparison of bindingproperties and biological activities of these VEGF-C proteins andmutants, using assays such as those described herein, will provide dataconcerning the significance of the observed major 32/29 kD and 21-23 kDVEGF-C processed forms. The data indicate that constructs encoding aminoacid residues 103-225 of the VEGF-C precursor (SEQ ID NO: 8) generate arecombinant ligand that is functional for both VEGFR-3 and VEGFR-2.

The data from this and preceding examples demonstrate that numerousfragments of the VEGF-C polypeptide retain biological activity. Anaturally occurring VEGF-C polypeptide spanning amino acids 103-226 (or103-227) of SEQ ID NO: 8, produced by a natural processing cleavagedefining the C-terminus, has been shown to be active. Example 27demonstrates that a fragment with residues 104-213 of SEQ ID NO: 8retains biological activity.

In addition, data from Example 21 demonstrates that a VEGF-C polypeptidehaving its amino terminus at position 112 of SEQ ID NO: 8 retainsactivity. Additional experiments have shown that a fragment lackingresidues 1-112 of SEQ ID NO: 8 retains biological activity.

In a related experiment, a stop codon was substituted for the lysine atposition 214 of SEQ ID NO: 8 (SEQ ID NO: 7, nucleotides 991-993). Theresulting recombinant polypeptide still was capable of inducing Flt4autophosphorylation, indicating that a polypeptide spanning amino acidresidues 113-213 of SEQ ID NO: 8 is biologically active.

Sequence comparisons of members of the VEGF family of polypeptidesprovides an indication that still smaller fragments of the polypeptidedepicted in SEQ ID NO: 8 will retain biological activity. In particular,eight highly conserved cysteine residues of the VEGF family ofpolypeptides define a region from residues 131-211 of SEQ ID NO: 8 (seeFIG. 10) of evolutionary significance; therefore, a polypeptide spanningfrom about residue 131 to about residue 211 is expected to retain VEGF-Cbiological activity. In fact, a polypeptide which retains the conservedmotif RCXXCC (e.g., a polypeptide comprising from about residue 161 toabout residue 211 of SEQ ID NO: 8 is postulated to retain VEGF-Cbiological activity. To maintain native conformation of these fragments,it may be preferred to retain about 1-2 additional amino acids at thecarboxy-terminus and 1-2 or more amino acids at the amino terminus.

Beyond the preceding considerations, evidence exists that smallerfragments and/or fragment analogs which lack the conserved cysteinesnonetheless will retain VEGF-C biological activity. Consequently, thematerials and methods of the invention include all VEGF-C fragments,variants, and analogs that retain at least one biological activity ofVEGF-C, regardless of the presence or absence of members of theconserved set of cysteine residues.

Example 29 Expression of Human VEGF-C Under the Human K14 KeratinPromoter in Transgenic Mice Induces Abundant Growth of Lymphatic Vesselsin the Skin

The Flt4 receptor tyrosine kinase is relatively specifically expressedin the endothelia of lymphatic vessels. Kaipainen et al., Proc. Natl.Acad. Sci. (USA), 92: 3566-3570 (1995). Furthermore, the VEGF-C growthfactor stimulates the Flt4 receptor, showing less activity towards theKDR receptor of blood vessels (Joukov et al., EMBO I, 15:290-298 (1996);See Example 26).

Experiments were conducted in transgenic mice to analyze the specificeffects of VEGF-C overexpression in tissues. The human K14 keratinpromoter is active in the basal cells of stratified squamous epithelia(Vassar et al., Proc. Natl. Acad. Sci. (USA), 86:1563-1567 (1989)) andwas used as the expression control element in the recombinant VEGF-Ctransgene. The vector containing the K14 keratin promoter is describedin Vassar et al., Genes Dev., 5:714-727 (1991) and Nelson et al., J.Cell Biol. 97:244-251 (1983).

The recombinant VEGF-C transgene was constructed using the human fulllength VEGF-C cDNA (GenBank Acc. No. X94216). This sequence was excisedfrom a pCI-neo vector (Promega) with XhoI/NotI, and the resulting 2027base pair fragment containing the open reading frame and stop codon(nucleotides 352-1611 of SEQ ID NO: 7) was isolated. The isolatedfragment was then subjected to an end-filling reaction using the Klenowfragment of DNA polymerase. The blunt-ended fragment was then ligated toa similarly opened BamHI restriction site in the K14 vector. Theresulting construct contained the EcoRI site derived from the polylinkerof the pCI-neo vector. This EcoRI site was removed using standardtechniques (a Klenow-mediated fill-in reaction following partialdigestion of the recombinant intermediate with EcoRI) to facilitate thesubsequent excision of the DNA fragment to be injected into fertilizedmouse oocytes. The resulting clone, designated K14-VEGF-C, isillustrated in FIG. 20 of commonly-owned PCT patent applicationPCT/FI96/00427, filed Aug. 1, 1996, published as WO 97/05250.

The EcoRI-HindIII fragment from clone K14 VEGF-C containing the K14promoter, VEGF-C cDNA, and K14 polyadenylation signal was isolated andinjected into fertilized oocytes of the FVB-NIH mouse strain. Theinjected zygotes were transplanted to oviducts of pseudopregnant C57BL/6XDBA/2J hybrid mice. The resulting founder mice were analyzed for thepresence of the transgene by polymerase chain reaction of tail DNA usingthe primers: 5′-CATGTACGAACCGCCAG-3′ (SEQ ID NO: 22) and5′-AATGACCAGAGAGAGGCGAG-3′ (SEQ ID NO: 23). In addition, the tail DNAswere subjected to EcoRV digestion and subsequent Southern analysis usingthe EcoRI-HindIII fragment injected into the mice. Out of 8 pupsanalyzed at 3 weeks of age, 2 were positive, having approximately 40-50copies and 4-6 copies of the transgene in their respective genomes.

The mouse with the high copy number transgene was small, developed moreslowly than its litter mates and had difficulty eating (i.e. suckling).Further examination showed a swollen, red snout and poor fur. Althoughfed with a special liquid diet, it suffered from edema of the upperrespiratory and digestive tracts after feeding and had breathingdifficulties. This mouse died eight weeks after birth and wasimmediately processed for histology, immunohistochemistry, and in situhybridization.

Histological examination showed that in comparison to the skin oflittermates, the dorsal dermis of K14-VEGF-C transgenic mice wasatrophic and connective tissue was replaced by large lacunae devoid ofred cells, but lined with a thin endothelial layer. These distendedvessel-like structures resembled those seen in human lymphangiomas. Thenumber of skin adnexal organs and hair follicles were reduced. In thesnout region, an increased number of vessels was also seen. Therefore,VEGF-C overexpression in the basal epidermis is capable of promoting thegrowth of extensive vessel structure in the underlying skin, includinglarge vessel lacunae. The endothelial cells surrounding these lacunaecontained abundant Flt4 mRNA in in situ hybridization (see Examples 23and 30 for methodology). The vessel morphology indicates that VEGF-Cstimulates the growth of vessels having features of lymphatic vessels.The other K14-VEGF-C transgenic mouse had a similar skin histopathology.

Nineteen additional pups were analyzed at 3 weeks of age for thepresence of the VEGF-C transgene, bring the number of analyzed pups totwenty-seven. A third transgene-positive pup was identified, havingapproximately 20 copies of the transgene in its genome. The 20 copymouse and the 4-6 copy mouse described above transmitted the gene to 6out of 11 and 2 out of 40 pups, respectively. The physiology of theseadditional transgenic mice were further analyzed.

The adult transgenic mice were small and had slightly swollen eyelidsand poorly developed fur. Histological examination showed that theepidermis was hyperplastic and the number of hair follicles was reduced;these effects were considered unspecific or secondary to otherphenotypic changes. The dermis was atrophic (45% of the dermalthickness, compared to 65% in littermate controls) and its connectivetissue was replaced by large dilated vessels devoid of red cells, butlined with a thin endothelial cell layer. Such abnormal vessels wereconfined to the dermis and resembled the dysfunctional, dilated spacescharacteristic of hyperplastic lymphatic vessels. See Fossum, et al., J.Vet. Int. Med., 6: 283-293 (1992). Also, the ultrastructural featureswere reminiscent of lymphatic vessels, which differ from blood vesselsby having overlapping endothelial junctions, anchoring filaments in thevessel wall, and a discontinuous or even partially absent basementmembrane. See Leak, Microvasc. Res., 2: 361-391 (1970). Furthermore,antibodies against collagen types IV, XVIII [Muragaki et al., Proc.Natl. Acad. Sci. USA, 92: 8763-8776 (1995)] and laminin gave very weakor no staining of the vessels, while the basement membrane staining ofother vessels was prominent. The endothelium was also characterized bypositive staining with monoclonal antibodies against desmoplakins I andII (Progen), expressed in lymphatic, but not in vascular endothelialcells. See Schmelz et al., Differentiation, 57: 97-117 (1994).Collectively, these findings strongly suggested that the abnormalvessels were of lymphatic origin.

In Northern hybridization studies, abundant VEGF-C mRNA was detected inthe epidermis and hair follicles of the transgenic mice, while mRNAsencoding its receptors VEGFR-3 and VEGFR-2 as well as the Tie-1endothelial receptor tyrosine kinase [Korhonen et al., Oncogene, 9:395-403 (1994)] were expressed in endothelial cells lining the abnormalvessels. In the skin of littermate control animals, VEGFR-3 could bedetected only in the superficial subpapillary layer of lymphaticvessels, while VEGFR-2 was found in all endothelia, in agreement withearlier findings. See Millauer et al., Cell, 72: 1-20 (1993); andKaipainen et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995).

The lymphatic endothelium has a great capacity to distend in order toadapt to its functional demands. To determine whether vessel dilationwas due to endothelial distension or proliferation, in vitroproliferation assays were conducted. Specifically, to measure DNAsynthesis, 3 mm×3 mm skin biopsies from four transgenic and four controlmice were incubated in D-MEM with 10 micrograms/ml BrdU for 6 hours at37° C., fixed in 70% ethanol for 12 hours, and embedded in paraffin.After a 30 minute treatment with 0.1% pepsin in 0.1 M HCl at roomtemperature to denature DNA, staining was performed using mousemonoclonal anti-BrdU antibodies (Amersham). It appeared that theVEGF-C-receptor interaction in the transgenic mice transduced amitogenic signal, because, in contrast to littermate controls, thelymphatic endothelium of the skin from young K14-VEGF-C mice showedincreased DNA synthesis as demonstrated by BrdU incorporation followedby staining with anti-BrdU antibodies. This data further confirms thatVEGF-C acts as a true growth factor in mammalian tissues.

In related experiments, a similar VEGF transgene did not inducelymphatic proliferation, but caused enhanced density of hyperpermeable,tortuous blood microvessels instead.

Angiogenesis is a multistep process which includes endothelialproliferation, sprouting, and migration. See Folkman et al., J. Biol.Chem., 267: 10931-10934 (1992). To estimate the contribution of suchprocesses to the transgenic phenotype, the morphology and function ofthe lymphatic vessels was analysed using fluorescent microlymphographyusing techniques known in the art. See Leu et al., Am. J. Physiol., 267:1507-1513 (1994); and Swartz et al., Am. J. Physiol., 270: 324-329(1996). Briefly, eight-week old mice were anesthetized and placed on aheating pad to maintain a 37° C. temperature. A 30-gauge needle,connected to a catheter filled with a solution of FITC-dextran 2M (8mg/ml in PBS), was injected intradermally into the tip of the tail. Thesolution was infused with a constant pressure of 50 cm water (averagingroughly 0.01 microliters per minute flow rate) until the extent ofnetwork filling remained constant (approximately 2 hours). Flow rate andfluorescence intensity were monitorerd continuously throughout theexperiment. In these experiments, a typical honeycomb-like network withsimilar mesh sizes was observed in both control and transgenic mice, butthe diameter of lymphatic vessels was about twice as large in thetransgenic mice, as summarized in the table below. (The intravitalfluorescence microscopy of blood vessels was performed as has beendescribed in the art. See Tukumura et al., Cancer Res., 55: 4824-4829(1995).)

Structural parameters of lymphatic and blood vessel networks transgeniccontrol P-value** (n = 4) (n = 5) lymphatic diameter 142.3 ± 26.2  68.2± 21.7 .0143 vessels* horizontal  1003 ± 87.1 960.8 ± 93.1 .2207 meshsize*** Vertical mesh 507.3 ± 58.9 488.8 ± 59.9 .5403 size (n = 3) (n =6) blood median  8.3 ± 0.6  7.6 ± 1.1 .1213 vessels diameter vesseldensity, 199.2 ± 6.6  216.4 ± 20.0 .3017 cm/cm² n = number of animals*mean (μm) ± SD **Mann-Whitney test ***mesh size describes vesseldensitySome dysfunction of the abnormal vessels was indicated by the fact thatit took longer for the dextran to completely fill the abnormal vessels.Injection of FITC-dextran into the tail vein, followed by fluorescencemicroscopy of the ear, showed that the blood vascular morphology wasunaltered and leukocyte rolling and adherence appeared normal in thetransgenic mice. These results suggest that the endothelialproliferation induced by VEGF-C leads to hyperplasia of the superficiallymphatic network but does not induce the sprouting of new vessels.

These effects of VEGF-C overexpression are unexpectedly specific,especially since, as described in other examples, VEGF-C is also capableof binding to and activating VEGFR-2, which is the major mitogenicreceptor of blood vessel endothelial cells. In culture, highconcentrations of VEGF-C stimulate the growth and migration of bovinecapillary endothelial cells which express VEGFR-2, but not significantamounts of VEGFR-3. In addition, VEGF-C induces vascular permeability inthe Miles assay [Miles, A. A., and Miles, E. M., J. Physiol.,118:228-257 (1952); and Udaka, et al., Proc. Soc. Exp. Biol. Med.,133:1384-1387 (1970)]; presumably via its effect on VEGFR-2. VEGF-C isless potent than VEGF in the Miles assay, 4- to 5-fold higherconcentrations of VEGF-C ΔNΔCHis being required to induce the samedegree of permeability. In vivo, the specific effects of VEGF-C onlymphatic endothelial cells may reflect a requirement for the formationof VEGFR-3×VEGFR-2 heterodimers for endothelial cell proliferation atphysiological concentrations of the growth factor. Such possibleheterodimers may help to explain how three homologous VEGFs exertpartially 10 redundant, yet strikingly specific biological effects.

The foregoing in vivo data indicates utilities for both (i) VEGF-Cpolypeptides and polypeptide variants and analogs having VEGF-Cbiological activity, and (ii) anti-VEGF-C antibodies and VEGF-Cantagonists that inhibit VEGF-C activity (e.g., by binding VEGF-C orinterfering with VEGF-C/receptor interactions. For example, the dataindicates a therapeutic utility for VEGF-C polypeptides in patientswherein growth of lymphatic tissue may be desirable (e.g., in patientsfollowing breast cancer or other surgery where lymphatic tissue has beenremoved and where lymphatic drainage has therefore been compromised,resulting in swelling; or in patients suffering from elephantiasis). Thedata indicates a therapeutic utility for anti-VEGF-C antibody substancesand VEGF-C antagonists for conditions wherein growth-inhibition oflymphatic tissue may be desirable (e.g., treatment of lymphangiomas).Accordingly, methods of administering VEGF-C and VEGF-C variants,analogs, and antagonists are contemplated as methods and materials ofthe invention.

Example 30 Expression of VEGF-C and Flt4 in the Developing Mouse

Embryos from a 16-day post-coitus pregnant mouse were prepared and fixedin 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 6μm. The sections were placed on silanated microscope slides and treatedwith xylene, rehydrated, fixed for 20 minutes in 4% PFA, treated withproteinase K (7 mg/ml; Merck, Darmstadt, Germany) for 5 minutes at roomtemperature, again fixed in 4% PFA and treated with acetic anhydride,dehydrated in solutions with increasing ethanol concentrations, driedand used for in situ hybridization.

In situ hybridization of sections was performed as described (Västrik etal., J. Cell Biol., 128:1197-1208 (1995)). A mouse VEGF-C antisense RNAprobe was generated from linearized pBluescript II SK+ plasmid(Stratagene Inc.), containing a fragment corresponding to nucleotides499-979 of mouse VEGF-C cDNA, where the noncoding region and the BR3Prepeat were removed by Exonuclease III treatment. The fragment had beencloned into the EcoRI and HindIII sites of pBluescript II SK+.Radiolabeled RNA was synthesized using T7 RNA Polymerase and [³⁵S]-UTP(Amersham, Little Chalfont, UK). About two million cpm of the VEGF-Cprobe was applied per slide. After an overnight hybridization, theslides were washed first in 2×SSC and 20-30 mM DDT for 1 hour at 50° C.Treatment continued with a high stringency wash, 4×SSC and 20 mM DTT and50% deionized formamide for 30 minutes at 65° C. followed by RNase Atreatment (20 μg/ml) for 30 minutes at 37° C. The high stringency washwas repeated for 45 minutes. Finally, the slides were dehydrated anddried for 30 minutes at room temperature. The slides were dipped intophotography emulsion and exposed for 4 weeks. Slides were developedusing Kodak D-16 developer, counterstained with hematoxylin and mountedwith Permount (FisherChemical).

For in situ hybridizations of Flt4 sequences, a mouse Flt4 cDNA fragmentcovering by 1-192 of the published sequence (Finnerty et al., Oncogene,8:2293-2298 (1993)) was used, and the above-described protocol wasfollowed, with the following exceptions. Approximately one million cpmof the Flt4 probe were applied to each slide. The stringent washesfollowing hybridization were performed in 1×SSC and 30 mM DTT for 105minutes.

Darkfield and lightfield photomicrographs from these experiments arepresented in commonly-owned PCT patent application PCTfFI96/00427, filedAug. 1, 1996, incorporated by reference herein. Observations from thephotomicrographs are summarized below.

The most prominently Flt4-hybridizing structures appeared to correspondto the developing lymphatic and venous endothelium. A plexus-likeendothelial vascular structure surrounding the developing nasopharyngealmucous membrane was observed. The most prominent signal using the VEGF-Cprobe was obtained from the posterior part of the developing nasalconchae, which in higher magnification showed the epithelium surroundingloose connective tissue/forming cartilage. This structure gave a strongin situ hybridization signal for VEGF-C. With the VEGF-C probe, moreweakly hybridizing areas were observed around the snout, although thissignal is much more homogeneous in appearance. Thus, the expression ofVEGF-C is strikingly high in the developing nasal to conchae.

The conchae are surrounded with a rich vascular plexus, important innasal physiology as a source for the mucus produced by the epithelialcells and for warming inhaled air. It is suggested that VEGF-C isimportant in the formation of the concheal venous plexus at the mucousmembranes, and that it may also regulate the permeability of the vesselsneeded for the secretion of nasal mucus. Possibly, VEGF-C and itsderivatives, and antagonists, could be used in the regulation of theturgor of the conchal tissue and mucous membranes and therefore thediameter of the upper respiratory tract, as well as the quantity andquality of mucus produced. These factors are of great clinicalsignificance in inflammatory (including allergic) and infectiousdiseases of the upper respiratory tract. Accordingly, the inventioncontemplates the use of the materials of the invention, includingVEGF-C, Flt4, and their derivatives, in methods of diagnosing andtreating inflammatory and infectious conditions affecting the upperrespiratory tract, including nasal structures.

Example 31 Characterization of the Exon-Intron Organization of the HumanVEGF-C Gene

Two genomic DNA clones covering exons 1, 2, and 3 of the human VEGF-Cgene were isolated from a human genomic DNA library using VEGF-C cDNAfragments as probes. In particular, a human genomic library inbacteriophage EMBL-3 lambda (Clontech) was screened using aPCR-generated fragment corresponding to nucleotides 629-746 of the humanVEGF-C cDNA (SEQ ID NO: 7). One positive clone, designated “lambda 3,”was identified, and the insert was subcloned as a 14 kb XhoI fragmentinto the pBluescript H (pBSK II) vector (Stratagene). The genomiclibrary also was screened with a labeled 130 by NotI-Sad fragment fromthe 5′-noncoding region of the VEGF-C cDNA (the NotI site is in thepolylinker of the cloning vector; the Sad site corresponds tonucleotides 92-97 of SEQ ID NO: 7). Two positive clones, designated“lambda 5” and “lambda 8,” were obtained. Restriction mapping analysisshowed that clone lambda 3 contains exons 2 and 3, while clone lambda 5contains exon 1 and the putative promoter region.

Three genomic fragments containing exons 4, 5, 6 and 7 were subclonedfrom a genomic VEGF-C P1 plasmid clone. In particular, purified DNA froma genomic P1 plasmid clone 7660 (Paavonen et al., Circulation, 93:1079-1082 (1996)) was used. EcoRI fragments of the P1 insert DNA wereligated into pBSK II vector. Subclones of clone 7660 which containedhuman VEGF-C cDNA homologous sequences were identified by colonyhybridization, using the full-length VEGF-C cDNA as a probe. Threedifferent genomic fragments were identified and isolated, whichcontained the remaining exons 4-7.

To determine the genomic organization, the clones were mapped usingrestriction endonuclease cleavage. Also, the coding regions andexon-intron junctions were partially sequenced. The result of thisanalysis is depicted in FIGS. 11A and 12. The sequences of allintron-exon boundaries (FIG. 11A, SEQ ID NOs: 24-35) conformed to theconsensus splicing signals (Mount, Nucl. Acids Res., 10: 459-472(1982)). The length of the intron between exon 5 and 6 was determineddirectly by nucleotide sequencing and found to be 301 bp. The length ofthe intron between exons 2 and 3 was determined by restriction mappingand Southern hybridization and was found to be about 1.6 kb. Each of theother introns is over 10 kb in length.

A similar analysis was performed for the murine genomic VEGF-C gene. Thesequences of murine VEGF-C intron-exon boundaries are depicted in FIG.11B and SEQ ID NOs: 36-47.

The restriction mapping and sequencing data indicated that the VEGF-Csignal sequence and the first residues of the N-terminal propeptide areencoded by exon 1. The second exon encodes the carboxy-terminal portionof the N-terminal propeptide and the amino terminus of the VEGF homologydomain. The most conserved sequences of the VEGF homology domain aredistributed in exons 3 (containing 6 conserved cysteine residues) and 4(containing 2 cys residues). The remaining exons encode cysteine-richmotifs of the type C-6X-C-10X-CRC (exons 5 and 7) and a fivefoldrepeated motif of type C-6X-B-3X-C-C-C, which is typical of a silkprotein.

To further characterize the human VEGF-C gene promoter, the lambda 5clone was further analyzed. Restriction mapping of this clone using acombination of single- and double-digestions and Southern hybridizationsindicated that it includes: (1) an approximately 6 kb region upstream ofthe putative initiator ATG codon, (2) exon 1, and (3) at least 5 kb ofintron I of the VEGF-C gene.

A 3.7 kb Xba I fragment of clone lambda 5, containing exon 1 and 5′ and3′ flanking sequences, was subcloned and further analyzed. As reportedpreviously, a major VEGF-C mRNA band migrates at a position of about 2.4kb. Calculating from the VEGF-C coding sequence of 1257 by and a 391 by3′ noncoding sequence plus a polyA sequence of about 50-200 bp, the mRNAstart site was estimated to be about 550-700 by upstream of thetranslation initiation codon.

RNase protection assays were employed to obtain a more preciselocalization of the mRNA start site. The results of these experimentsindicated that the RNA start site in the human VEGF-C gene is located539 by upstream of the ATG translational initiation codon.

To further characterize the promoter of the human VEGF-C gene, a genomicclone encompassing about 2.4 kb upstream of the translation initiationsite was isolated, and the 5′ noncoding cDNA sequence and putativepromoter region were sequenced. The sequence obtained is set forth inSEQ ID NO: 48. (The beginning of the VEGF-C cDNA sequence set forth inSEQ ID NO: 7 corresponds to position 2632 of SEQ ID NO: 48; thetranslation initiation codon corresponds to positions 2983-2985 of SEQID NO: 48.) Similar to what has been observed with the VEGF gene, theVEGF-C promoter is rich in G and C residues and lacks consensus TATA andCCAAT sequences. Instead, it has numerous putative binding sites(5′-GGGCGG-3′ or 5′-CCGCCC-3′) for Sp1, a ubiquitous nuclear proteinthat can initiate transcription of TATA-less genes. See Pugh and Tjian,Genes and Dev., 5:105-119 (1991). In addition, sequences upstream of theVEGF-C translation start site were found to contain frequent consensusbinding sites for the AP-2 factor (5′-GCCN₃GCC-3′) and binding sites forthe AP-1 factor (5′-TKASTCA-3′). Binding sites for regulators oftissue-specific gene expression, like NFkB and GATA, are located in thedistant part of VEGF-C promoter. This suggests that the cAMP-dependentprotein kinase and protein kinase C, as activators of AP-2 transcriptionfactor [Curran and Franza, Cell, 55:395-397 (1988)], mediate VEGF-Ctranscriptional regulation.

The VEGF-C gene is abundantly expressed in adult human tissues, such asheart, placenta, ovary and small intestine, and is induced by a varietyof factors. Indeed, several potential binding sites for regulators oftissue-specific gene expression, like NFkB (5′-GGGRNTYYC-3′) and GATA,are located in the distal part of the VEGF-C promoter. For example, NFkBis known to regulate the expression of tissue factor in endothelialcells. Also, transcription factors of the GATA family are thought toregulate cell-type specific gene expression.

Unlike VEGF, the VEGF-C gene does not contain a binding site for thehypoxia-inducible factor, HIF-1 (Levy et al., J. Biol. Chem., 270:13333-13340 (1995)). This finding suggests that if the VEGF-C mRNA isregulated by hypoxia, the mechanism would be based mainly on theregulation of mRNA stability. In this regard, numerous studies haveshown that the major control point for the hypoxic induction of the VEGFgene is the regulation of the steady-state level of mRNA. See Levy etal., J. Biol. Chem., 271: 2746-2753 (1996). The relative rate of VEGFmRNA stability and decay is considered to be determined by the presenceof specific sequence motifs in its 3′ untranslated region (UTR), whichhave been demonstrated to regulate mRNA stability. (Chen and Shyu, Mol.Cell. Biol., 14: 8471-8482 (1994)). The 3′-UTR of the VEGF-C gene alsocontains a putative motif of this type (TTATTT), at positions 1873-1878of SEQ ID NO: 7.

To identify DNA elements important for basal expression of VEGF-C intransfected cells, a set of luciferase reporter plasmids containingserial 5′ deletions through the promoter region was constructed.Restriction fragments of genomic DNA containing 5′ portions of the firstexon were cloned into the polylinker of the pGL3 reporter vector(Promega) and confirmed by sequencing. About 10 μg of the individualconstructs in combination with 2 μg of pSV2-β-galactosidase plasmid(used as a control of transfection efficiency) were transfected intoHeLa cells using the calcium phosphate-mediated transfection method. Twodays after transfection, the cells were harvested and subjected to theluciferase assay. The luciferase activity was normalized to that of thepGL3 control vector driven by SV40 promoter/enhancer.

As depicted in FIG. 3, the 5.5 kb XhoI-RsrII fragment of clone lambda 5gave nearly 9-fold elevated activity when compared with a promoterlessvector. Deletion of a 5′ XhoI-HindIII fragment of 2 kb had no effect onthe promoter activity. The activity of the 1.16 kb XbaI-RsrII fragmentwas about twice that of the pGL3 basic vector, while the activity of thesame fragment in the reverse orientation was at background level.Further deletion of the XbaI-SacI fragment caused an increase in thepromoter activity, suggesting the presence of silencer elements in theregion from −1057 to −199 (i.e., 199 to 1057 by upstream from thetranscription initiation site). The shortest fragment (SacII-RsrII)yielded only background activity, which was consistent with the factthat the mRNA initiation site was not present in this construct.

To determine whether further sequences in the first exon of human VEGF-Care important for basal expression, an RsrII fragment spanningnucleotides 214-495 (i.e., 214-495 by downstream from the transcriptioninitiation site) was subcloned in between of XbaI-RsrII fragment and theluciferase reporter gene. Indeed, the obtained construct showed an 50%increase in activity when compared with the XbaI-RsrII construct.

The VEGF gene has been shown to be up-regulated by a number of stimuliincluding serum derived growth factors. To find out whether the VEGF-Cgene also can be stimulated by serum, RNA from serum-starved andserum-stimulated HT1080 cells was subjected to primer extensionanalysis, which demonstrated that VEGF-C mRNA is up-regulated by serumstimulation.

Additional serum stimulation experiments indicated that the serumstimulation leads to increased VEGF-C promoter activity. Cells weretransfected as described above and 24 h after transfection changed intomedium containing 0.5% bovine serum albumin. Cells were then stimulatedwith 10% fetal calf serum for 4 hours and analyzed. The XbaI-RsrIIpromoter construct derived from lambda 5 yielded a twofold increasedactivity upon serum stimulation, while the same fragment in the reverseorientation showed no response. All other promoter constructs alsoshowed up-regulation, ranging from 1.4 to 1.6 fold (FIG. 3).

Example 32 Identification of a VEGF-C Splice Variant

As reported in Example 16, a major 2.4 kb VEGF-C mRNA and smalleramounts of a 2.0 kb mRNA are observable. To clarify the origin of theseRNAs, several additional VEGF-C cDNAs were isolated and characterized. Ahuman fibrosarcoma cDNA library from HT1080 cells in the lambda gt11vector (Clontech, product #HL1048b) was screened using a 153 by humanVEGF-C cDNA fragment as a probe as described in Example 10. See alsoJoukov et al., EMBO J., 15:290-298 (1996). Nine positive clones werepicked and analyzed by PCR amplification using oligonucleotides5′-CACGGCTTATGCAAGCAAAG-3′ (SEQ ID NO: 49) and5′-AACACAGTTTTCCATAATAG-3′ (SEQ ID NO: 50) These oligonucleotides wereselected to amplify the portion of the VEGF-C cDNA corresponding tonucleotides 495-1661 of SEQ ID NO: 7. PCR was performed using anannealing temperature of 55° C. and 25 cycles.

The resultant PCR products were electrophoresed on agarose gels. Fiveclones out of the nine analyzed generated PCR fragments of the expectedlength of 1147 base pairs, whereas one was slightly shorter. The shorterfragment and one of the fragments of expected length were cloned intothe pCRTMII vector (Invitrogen) and analyzed by sequencing. The sequencerevealed that the shorter PCR fragment had a deletion of 153 base pairs,corresponding to nucleotides 904 to 1055 of SEQ ID NO: 7. These deletedbases correspond to exon 4 of the human and mouse VEGF-C genes,schematically depicted in FIGS. 13A and 13B. Deletion of exon 4 resultsin a frameshift, which in turn results in a C-terminal truncation of thefull-length VEGF-C precursor, with fifteen amino acid residuestranslated from exon 5 in a different frame than the frame used toexpress the full-length protein. Thus, the C-terminal amino acidsequence of the resulting truncated polypeptide would be -Leu(181)-Ser-Lys-Thr-Val-Ser-Gly-Ser-Glu-Gln-Asp-Leu-Pro-His-Glu-Leu-His-Val-Glu(199)(SEQ ID NO: 51). The polypeptide encoded by this splice variant wouldnot contain the C-terminal cleavage site of the VEGF-C precursor. Thus,a putative alternatively spliced RNA form lacking conserved exon 4 wasidentified in HT-1080 fibrosarcoma cells and this form is predicted toencode a protein of 199 amino acid residues, which could be anantagonist of VEGF-C.

Example 33 VEGF-C is Similarly Processed in Different Cell Cultures InVitro

To study whether VEGF-C is similarly processed in different cell types,293 EBNA cells, COS-1 cells and HT-1080 cells were transfected with wildtype human VEGF-C cDNA and labelled with Pro-Mix™ as described inExample 22. The conditioned media from the cultures were collected andsubjected to immunoprecipitation using antiserum 882 (described inExample 21, recognizing a peptide corresponding to amino acids 104-120of SEQ ID NO: 8). The immunoprecipitated polypeptides were separated viaSDS-PAGE, and detected via autoradiography. The major form of secretedrecombinant VEGF-C observed from all cell lines tested is a 29/32 kDdoublet. These two polypeptides are bound to each other by disulfidebonds, as described in Example 22. A less prominent band ofapproximately 21 kD also was detected in the culture media.Additionally, a non-processed VEGF-C precursor of 63 kDa was observed.This form was more prominent in the COS-1 cells, suggesting thatproteolytic processing of VEGF-C in COS cells is less efficient than in293 EBNA cells. Endogenous VEGF-C (in non-transfected cells) was notdetectable under these experimental conditions in the HT-1080 cells, butwas readily detected in the conditioned medium of the PC-3 cells.Analysis of the subunit polypeptide sizes and ratios in PC-3 cells and293 EBNA cells revealed strikingly similar results: the most prominentform was a doublet of 29/32 kDa and a less prominent form the 21 kDpolypeptide. The 21 kD form produced by 293 EBNA cells was notrecognized by the 882 antibody in the Western blot, although it isrecognized when the same antibody is used for immunoprecipitation (seedata in previous examples). As reported in Example 21, cleavage of the32 kD form in 293 EBNA cells occurs between amino acid residues 111 and112 (SEQ ID NO: 8), downstream of the cleavage site in PC-3 cells(between residues 102 and 103). Therefore, the 21 kD form produced in293 EBNA cells does not contain the complete N-terminal peptide used togenerate antiserum 882. In a related experiment, PC-3 cells werecultured in serum-free medium for varying periods of time (1-8 days)prior to isolation of the conditioned medium. The conditioned medium wasconcentrated using a Centricon device (Amicon, Beverly, USA) andsubjected to Western blotting analysis using antiserum 882. After oneday of culturing, a prominent 32 kD band was detected. Increasingamounts of a 21-23 kD form were detected in the conditioned media from 4day and 8 day cultures. The diffuse nature of this polypeptide band,which is simply called the 23 kD polypeptide in example 5 and severalsubsequent examples, is most likely due to a heterogenous and variableamount of glycosylation. These results indicate that, initially, thecells secrete a 32 kD polypeptide, which is further processed or cleavedin the medium to yield the 21-23 kD form. The microheterogeneity of thispolypeptide band would then arise from the variable glycosylation degreeand, from microheterogeneity of the processing cleavage sites, such asobtained for the amino terminus in PC-3 and 293 EBNA cell cultures. Thecarboxyl terminal cleavage site could also vary, examples of possiblecleavage sites would be between residues 225-226, 226-227 and 227-228 aswell as between residues 216-217. Taken together, these data suggest thepossibility that secreted cellular protease(s) are responsible for thegeneration of the 21-23 kD form of VEGF-C from the 32 kD polypeptide.Such proteases could be used in vitro to cleave VEGF-C precursorproteins in solution during the production of VEGF-C, or used in cellculture and in vivo to release biologically active VEGF-C.

Example 34 Differential Binding of VEGF-C Forms by the ExtracellularDomains of VEGFR-3 and VEGFR-2

In two parallel experiments, 293 EBNA cells were transfected with aconstruct encoding recombinant wild type VEGF-C or a construct encodingVEGF-C ΔNΔCHis (Example 28) and about 48 hours after transfection,metabolically labelled with Pro-Mix™ as described in previous examples.The media were collected from mock-transfected and transfected cells andused for receptor binding analyses.

Receptor binding was carried out in binding buffer (PBS, 0.5% BSA; 0.02%Tween 20, 1 microgram/ml heparin) containing approximately 0.2 microgramof either (a) a fusion protein comprising a VEGFR-3 extracellular domainfused to an immunoglobulin sequence (VEGFR-3-Ig) or (b) a fusion proteincomprising VEGFR-2 extracellular domain fused to an alkaline phosphatasesequence (VEGFR-2-AP; Cao et al., J. Biol. Chem. 271:3154-62 (1996)). Asa control, similar aliquots of the 293 EBNA conditioned media were mixedwith 2 μl of anti-VEGF-C antiserum (VEGF-C IP).

After incubation for 2 hours at room temperature, anti-VEGF-C antibodiesand VEGFR-3-Ig protein were adsorbed to protein A-sepharose (PAS) andVEGFR-2-AP was immunoprecipitated using anti-AP monoclonal antibodies(Medix Biotech, Genzyme Diagnostics, San Carlos, Calif., USA) andprotein G-sepharose. Complexes containing VEGF-C bound to VEGFR-3-Ig orVEGFR-2-AP were washed three times in binding buffer, twice in 20 mMTris-HCl (pH 7.4) and VEGF-C immunoprecipitates were washed three timesin RIPA buffer and twice in 20 mM tris-HCl (pH 7.4) and analyzed viaSDS-PAGE under reducing and nonreducing conditions. As a control, thesame media were precipitated with antiAP and protein G-sepharose (PGS)or with PAS to control for possible nonspecific adsorption.

These experiments revealed that VEGFR-3 bound to both the 32/29 kD and21-23 kD forms of recombinant VEGF-C, whereas VEGFR-2 boundpreferentially to the 21-23 kD component from the conditioned media. Inaddition, small amounts of 63 kD and 52 kD VEGF-C forms were observedbinding with VEGFR-3. Further analysis under nonreducing conditionsindicates that a great proportion of the 21-23 kD VEGF-C bound to eitherreceptor does not contain interchain disulfide bonds. These findingsreinforce the results that VEGF-C binds VEGFR-2. This data suggests autility for recombinant forms of VEGF-C which are active towards VEGFR-3only or which are active towards both VEGFR-3 and VEGFR-2. On the otherhand, these results, together with the results in Example 28, do noteliminate the possibility that the 32/29 kD dimer binds VEGFR-3 but doesnot activate it. The failure of the 32/29 kD dimer to activate VEGFR-3could explain the finding that conditioned medium from the N-His VEGF-Ctransfected cells induced a less prominent tyrosine phosphorylation ofVEGFR-3 than medium from VEGF-C ΔNΔCHis transfected cells, even thoughexpression of the former polypeptide was much higher. Stable VEGF-Cpolypeptide mutants that bind to a VEGF-C receptor but fail to activatethe receptor are useful as VEGF-C antagonists.

Example 35 Discovery of VEGF-C Analogs that Selectively Bind to andActivate VEGFR-3, but not VEGFR-2

To further identify the cysteine residues of VEGF-C that are criticalfor retaining VEGF-C biological activities, an additional VEGF-C mutant,designated VEGF-CΔNΔCH is C156S, was synthesized, in which the cysteineresidue at position 156 of the 419 amino acid VEGF-C precursor (SEQ IDNO: 8; Genbank accession number X94216) was replaced with a serineresidue.

The mutagenesis procedure was carried out using the construct ofVEGF-CΔNΔCHis (see Example 28), cloned in the pALTER vector, and theAltered sites II in vitro mutagenesis system of Promega. Anoligonucleotide 5′-GACGGACACAGATGGAGGTTTAAAG-3′ (SEQ ID NO: 52) was usedto introduce the desired mutation in the cDNA encoding VEGF-CΔNΔCHis.The resulting mutated VEGF-C cDNA fragment was subcloned into theHindIII/NotI sites of the pREP-7 vector (Invitrogen), and the finalconstruct was re-sequenced to confirm the C156S mutation. The resultantclone has an open reading frame encoding amino acids 103-225 of SEQ IDNO: 8 (with a serine codon at position 156), and further encoding a6×His tag.

The wildtype VEGF-C cDNA and three VEGF-C mutant constructs (VEGF-CR226,227S, VEGF-C ΔNΔCHis, and VEGF-C ΔNΔCH is C156S) were used totransfect 293 EBNA cells, which were subcultured 16 hours aftertransfection. About 48 hours after transfection, the media were changedto DMEM/0.1% BSA, and incubation in this medium was continued for anadditional 48 hours. The resultant conditioned media were concentrated30-fold using Centriprep-10 (Amicon), and the amount of VEGF-C in themedia was analyzed by Western blotting using the anti-VEGF-C antiserum882 for immunodetection. Different amounts of the recombinant VEGF-CΔNΔCHis, purified from a yeast expression system, were analyzed inparallel as reference samples to measure and equalize the VEGF-Cconcentrations in the conditioned media. The conditioned medium frommock-transfected cells was used to dilute the VEGF-C conditioned mediato achieve equal concentrations.

An aliquot of the transfected cells were metabolically labelled for 6hours with 100 microcuries/ml of the PRO-MIX™ L-[³⁵S] in vitro celllabelling mix (Amersham). The conditioned media were collected, andbinding of the radioactively labelled VEGF-C proteins to theextracellular domains of VEGFR-3 and VEGFR-2 was analyzed usingrecombinantly produced VEGFR-3EC-Ig and VEGFR-2EC-Ig constructs(containing seven and three Ig loops of the extracellular domains of therespective receptors, fused to an immunoglobulin heavy chain constantregion).

All processed VEGF-C forms secreted to the culture medium bound toVEGFR-3EC domain, with preferential binding of the 21 kDa form. Whenpresent at high concentrations, the VEGF-C forms of 58 kDa and 29/31 kDabound to some extent non-specifically to protein A Sepharose.

The VEGFR-2EC domain preferentially bound the mature 21 kDa form ofwildtype VEGF-C and VEGF-CΔNΔCHis. Significantly, VEGF-CΔNΔCH is C156Sfailed to bind the VEGFR2-EC.

Next, the ability of the above-described VEGF-C polypeptides to competewith the ¹²⁵I-VEGF-CΔNΔCHis for binding to VEGFR-2 and VEGFR-3 wasanalyzed. Scatchard analysis using VEGF-C ΔCΔNHis provided indicationsof the VEGF-C binding affinity for VEGFR-3 (K_(D)=135 pM) and VEGFR-2(K_(D)=410 pM). Ten micrograms of the purified yeast VEGF-C ΔNΔCHis waslabeled using 3 mCi of Iodine-125, carrier-free (Amersham), and anIodo-Gen Iodination Reagent (Pierce), according to the standard protocolof Pierce. The resulting specific activity of the labeled VEGF-CΔNΔCHiswas 1.25×10⁵ cpm/ng.

To study receptor binding, PAE/VEGFR-2 and PAE/VEGFR-3 cells were seededinto 24-well tissue culture plates (Nunclon), which had been coated with2% gelatin in PBS. The ¹²⁵I-VEGF-C ΔNΔCHis (2×10⁵ cpm) and differentamounts of media containing equal concentrations of the non-labeledVEGF-C (wildtype and mutants) were added to each plate in Ham's F12medium, containing 25 mM HEPES (pH 8.0), 0.1% BSA, and 0.1% NaN₃. Thebinding was allowed to proceed at room temperature for 90 minutes. Theplates were then transferred onto ice and washed three times withice-cold PBS containing 0.1% BSA. The cells were then lysed in 1 M NaOH,the lysates were collected, and the radioactivity was measured using aγ-counter. Binding in the presence of VEGF-C-containing conditionedmedium was calculated as a percentage of binding observed in parallelcontrol studies wherein equal volumes of medium from mock-transfectedcells were used instead of VEGF-C conditioned media.

As shown in FIG. 4, left panel, all VEGF-C mutants displaced¹²⁵I-VEGF-CΔNΔCHis from VEGFR-3. The efficiency of displacement was asfollows: VEGF-CΔNΔCHisC156S>VEGF-CΔNΔCHis>wildtypeVEGF-C>VEGF-CR226,227S. These results indicate that enhanced binding toVEGFR-3 was obtained upon “recombinant maturation” of VEGF-C.Recombinant VEGF165 failed to displace VEGF-C from VEGFR-3.

VEGF, VEGF-CΔNΔCHis, and wildtype VEGF-C all efficiently displacedlabeled VEGF-CΔNΔCHis from VEGFR-2, with VEGF-CΔNΔCHis being more potentwhen compared to wildtype VEGF-C (FIG. 4, right panel). Thenon-processed VEGF-C R226,227S showed only weak competition of¹²⁵I-VEGF-CΔNΔCHis.

Surprisingly, VEGF-CΔNΔCHis R156S failed to displace VEGF-CΔNΔCHis fromVEGFR-2, thus confirming the above described results obtained using asoluble extracellular domain of VEGFR-2.

The ability of the above mentioned VEGF-C forms to stimulate tyrosinephosphorylation of VEGFR-3 and VEGFR-2 was also investigated.Importantly, identical dilutions of the conditioned media were used forthese experiments and for the competitive binding experiments describedabove. A Western blot analysis of the conditioned media usinganti-VEGF-C antiserum 882 was performed to confirm the approximatelyequal relative amounts of the factors present.

The stimulation of VEGFR-3 and VEGFR-2 autophosphorylation by thedifferent VEGF-C forms in general correlated with their bindingproperties, as well as with the degree of “recombinant processing” ofVEGF-C. The VEGF-CΔNΔCHisC156S appeared to be at least as potent asVEGF-CΔNΔCHis in stimulating VEGFR-3 autophosphorylation. VEGF-CΔNΔCHisshowed a higher potency when compared to wildtype VEGF-C in its abilityto stimulate tyrosine autophosphorylation of both VEGFR-2 and VEGFR-3.The VEGF-CR226,227S conditioned medium possessed a considerably weakereffect on autophosphorylation of VEGFR-3, and almost no effect onVEGFR-2 autophosphorylation.

Stimulation of VEGFR-2 tyrosine phosphorylation by VEGF-CΔNΔCH is C156Sdid not differ from that of conditioned medium from the mock transfectedcells, thus confirming the lack of VEGFR-2-binding andVEGFR-2-activating properties of this mutant.

The ability of VEGF-C ΔNΔCH is C156S to alter vascular permeability invivo was analyzed using the Miles assay (see Example 29). Therecombinant VEGF-C forms assayed (ΔNΔCHis, ΔNΔCH is C156S) were producedby 293 cells, purified from conditioned media using Ni-NTA Superflowresin (QIAGEN) as previously described, and pretreated with 15 μg/ml ofanti-human VEGF neutralizing antibody (R&D systems) to neutralizeresidual amounts of co-purified, endogenously produced VEGF. Eightpicomoles of the various VEGF-C forms, as well as 2 pmol of recombinanthuman VEGF165 (R&D systems) and approximately 2 pmol of VEGF165 from theconditioned medium which were either non-treated or pretreated with theabove mentioned VEGF-neutralizing antibody were injected subcutaneouslyto the back region of a guinea pig. The area of injection was analyzed20 minutes after injections. Both VEGF and VEGF-C ΔNΔCHis causedincreases in vascular permeability, whereas ΔNΔCH is C156S did notaffect vascular permeability. The neutralizing antibody completelyblocked permeability activity of VEGF but did not affect VEGF-Cactivity. Residual permeability activity observed for theVEGF-containing conditioned medium even after its treatment with VEGFneutralizing antibody was presumably caused by permeability factorsother than VEGF that are produced by 293 cells.

In yet another assay, the ability of VEGF-CΔNΔCHis and VEGF-CΔNΔCH isC156S to stimulate migration of bovine capillary endothelial cells in acollagen gel was analyzed. The ΔNΔCHis form dose-dependently stimulatedmigration, whereas the ΔNΔCH is C156S form had no significant activityin the assay.

The Miles assay also was used to assay the ability of VEGF-C R226,227S(8 pM, pretreated with anti-VEGF antibody) to induce vascularpermeability. The results indicated that the ability of VEGF-C R226,227Sto induce vascular permeability was much weaker when compared towildtype and ΔNΔCHis forms of VEGF-C. Collectively, this Miles assaydata is consistent with the VEGFR-2 binding and autophosphorylation datadescribed above, and indicates that VEGF-C effect on vascularpermeability is mediated via VEGFR-2.

Mitogenic signals from growth factor receptors are frequently relayedvia the extracellular signal regulated kinases/mitogen activated proteinkinases (ERK/MAPK) pathway into the nucleus. Purified recombinantVEGF-CΔNΔCHis and VEGF-C ΔNΔC156S produced by a Pichia expression systemwere used to determine MAPK pathway activation of cells expressingeither VEGFR-2 or VEGFR-3. The growth factor treated cells were lysed,and activated MAPK was detected using Western blotting with antibodiesagainst the phosphorylated forms of ERK1 and ERK2. At a concentration of100 ng/ml, VEGF-CΔNΔCHis showed rapid activation of the ERK1 and ERK2MAPK in both VEGFR-2- and VEGFR-3-expressing cells. In contrast,VEGF-CΔNΔC156S activated ERK1 and ERK2 exclusively in theVEGFR-3-expressing cells. At the concentrations used, both VEGF-CΔNΔCHisand VEGF-C ΔNΔC156S appeared to be equally potent in activating the MAPKthrough VEGFR-3. The amounts of total MAPK protein were confirmed to besimilar in the treated and untreated cells, as shown by staining of thefilter with p44/p42 MAPK antibodies made against a synthetic peptide ofrat p42.

The foregoing data indicates that proteolytic processing of VEGF-Cresults in an increase in its ability to bind and to activate VEGFR-3and VEGFR-2. Non-processed VEGF-C is a ligand and an activator ofpreferentially VEGFR-3, while the mature 21/23 kDa VEGF-C form is a highaffinity ligand and an activator of both VEGFR-3 and VEGFR-2.

Moreover, replacement of the cysteine residue at position 156 (ofprepro-VEGF-C, SEQ ID NO: 8) creates a selective ligand and activator ofVEGFR-3. This alteration inactivates the ability of processed VEGF-C tobind to VEGFR-2 and to activate VEGFR-2. Importantly, it is believedthat the elimination of the cysteine at position 156 is the alterationresponsible for this unexpected alteration in VEGF-C selectivity, andnot the substitution of a serine per se. It is expected that replacementof the cysteine at position 156 with other amino acids, or the meredeletion of this cysteine, will also result in VEGF-C analogs havingselective biological activity with respect to VEGFR-3. All suchreplacement and deletion analogs (collectively referred to as VEGF-CΔC₁₅₆ polypeptides) are contemplated as aspects of the presentinvention. Thus, “VEGF-C ΔC₁₅₆ polypeptides” of the invention derivedfrom human VEGF-C include polypeptides depicted in SEQ ID NO: 58,fragments of those polypeptides (especially fragments having an aminoterminus anywhere between residues 102 and 161 of SEQ ID NO: 58 and acarboxy-terminus anywhere between residues 210 and 228 of SEQ ID NO:58). “VEGF-C ΔC₁₅₆ polypeptides” of the invention also include thecorresponding polypeptides derived from murine, quail, and otherwildtype VEGF-C polypeptides.

VEGF-C polypeptides that have the C156S mutation (or functionallyequivalent mutations at position 156) and that retain biologicalactivity with respect to VEGFR-3, such as VEGF-C ΔNΔCH is C156S, areuseful in all of the same manners described above for wildtype VEGF-Cproteins and biologically active fragments thereof where VEGFR-3stimulation is desired. It is contemplated that most biologically activeVEGF-C fragments and processing variants, including but not limited tothe biologically active fragments and variants identified in precedingexamples, will retain VEGF-C biological activity (as mediated throughVEGFR-3) when a ΔC₁₅₆ mutation is introduced. All such biologicallyactive VEGF-C ΔC₁₅₆ polypeptides are intended as an aspect of thepresent invention.

Moreover, VEGF-C forms containing the C156S mutation or equivalentmutations can be used to distinguish those effects of VEGF-C mediatedvia VEGFR-3 and VEGFR-2 from those obtained via only VEGFR-3. Theability of such VEGF-C polypeptides to selectively stimulate VEGFR-3 arealso expected to be useful in clinical practice, it being understoodthat selectivity of a pharmaceutical is highly desirable in manyclinical contexts. For example, the selectivity of VEGF-C ΔC₁₅₆polypeptides for VEGFR-3 binding suggests a utility for these peptidesto modulate VEGF-C biological activities mediated through VEGFR-3,without significant concomitant modulation of blood vessel permeabilityor other VEGF-C activities that are modulated through VEGFR-2.

The data presented herein also indicates a utility for ΔC₁₅₆polypeptides that are capable of binding VEGFR-3, but that do not retainbiological activity mediated through VEGFR-3. Specifically, such formsare believed to be capable of competing with wildtype VEGF-C for bindingto VEGFR-3, and are therefore contemplated as molecules that inhibitVEGF-C-mediated stimulation of VEGFR-3. Because of the ΔC₁₅₆ alteration,such polypeptides (especially covalent or noncovalent dimers of suchpolypeptides) are not expected to bind VEGFR-2. Thus, certain ΔC₁₅₆polypeptides and polypeptide dimers are expected to have utility asselective inhibitors of VEGF-C biological activity mediated throughVEGFR-3 (i.e., without substantially altering VEGF-C mediatedstimulation of VEGFR-2).

In another embodiment of the invention, heterodimers comprising abiologically active VEGF-C polypeptide in association with a ΔC₁₅₆polypeptide are contemplated. It is contemplated that such heterodimerscan be formed in vitro, as described below in Example 37, or formed invivo with endogenous VEGF-C following administration of a ΔC₁₅₆polypeptide. Such heterodimers are contemplated as modulators of VEGF-Cmediated effects in cells where the biological effects of VEGF-C aremediated through VEGFR-2NEGFR-3 heterodimers. VEGF-C ΔC₁₅₆ polypeptidesin homodimers or in heterodimers with wt VEGF-C might selectivelyinhibit the ability of the latter to induce VEGF-like effects,particularly to increase the vascular permeability.

Replacement of the second and/or the fourth of the eight conservedcysteine residues of VEGF abolishes VEGF dimer formation and VEGFbiological activity. The analogous effect was investigated for VEGF-C,wherein the cysteines at positions 156 and 165 of SEQ ID NO: 8correspond to the second and fourth conserved cysteines. No homodimerswere obtained when VEGF-CΔNΔCH is C156,1658 (i.e., Cys₁₅₆ and Cys₁₆₅both replaced with serine residues) or in VEGF-CΔNΔCH is C1658 werechemically crosslinked. On the other hand, about half of bothcrosslinked VEGF-CΔNΔCHis and VEGF-CΔNΔCH is C1568 migrated as dimers.This data indicates that VEGF-CΔNΔCH is C1568 forms homodimers.Moreover, unlike VEGF-CΔNΔCHis, which forms preferentiallynon-covalently bound dimers, a fraction of VEGF-CΔNΔCH is C1568 wasdisulfide bonded, as detected by SDS-PAGE in non-reducing conditions. Inreceptor binding studies (using procedures such as those describedabove), the C165S and C156,165S forms were both unable to bind VEGFR-3or VEGFR-2. Collectively, these data suggest that homodimerization isrequired for VEGFR-3 activation by VEGF-C, and indicate that theinability of ΔNΔC156S to activate VEGFR-2 and to induce VEGF-likeeffects is not due to an inability of this mutant to form homodimers.

Example 36 Utility for VEGF-C in Promoting Myelopoiesis

The effects of VEGF-C on hematopoiesis were also analyzed. Specifically,leukocytes populations were analyzed in blood samples taken from the F1transgenic mice described in Example 29, and from their non-transgeniclittermates. Leukocyte population data from these mice and fromnon-transgenic FVB-NIH control mice (i.e., the strain used to generatethe transgenic mice) are set forth in the tables below.

FVB/NIH MICE male male female male 5.5 5.5 9.5 9.5 Cell Type monthsmonths months months mean ± σ Lymphocytes 72.20% 82.17% 84.25% 74.25%78.22 ± 5.10  Neutrophils 23.00% 15.17% 14.25% 22.25% 18.67 ± 3.98 Monocytes 0.65% 1.00% 0.25% 0.50% 0.60 ± 0.27 Eosinophils 2.15% 1.70%1.25% 3.00% 2.03 ± 0.65 Basophils 0.00% 0.00% 0.00% 0.00% 0 ± 0 VEGF-CTRANSGENIC MICE male male male Cell Type 2 months 3.5 months 7 monthsmean ± σ Lymphocytes 41.3% 41.50% 18.70% 33.83 ± 10.70 Neutrophils 55.3%53.80% 80.17% 63.09 ± 12.09 Monocytes 2.16% 1.30% 0.67% 1.38 ± 0.61Eosinophils 1.17% 3.50% .50% 1.72 ± 1.29 Basophils 0.00% 0.00% 0.00% 0 ±0 VEGF-C NEGATIVE CONTROL MICE (NON-TRANSGENIC LITTERMATES OF VEGF-CTRANSGENIC MICE) male male male male 2 3.5 7 Cell Type 2 months monthsmonths months mean ± σ Lymphocytes 89.00% 67.50% 91.00% 71.30%  79.7 ±10.41 Neutrophils 7.75% 23.00% 7.00% 23.75% 15.38 ± 8.01  Monocytes1.50% 0.50% 0.83% 0.75% 0.90 ± 0.37 Eosinophils 1.50% 9.00% 0.67% 4.00%3.79 ± 3.25 Basophils 0.00% 0.00% 0.50% 0.50% 0.25 ± 0.25As the foregoing data indicates, the overexpression of VEGF-C in theskin of the to transgenic mice correlates with a distinct alteration inleukocyte populations. Notably, the measured populations of neutrophilswere markedly increased in the transgenic mice. One explanation for themarked increase in neutrophils is a myelopoietic activity attributableto VEGF-C. A VEGF-C influence on leukocyte trafficking in and out oftissues also may effect observed neutrophil populations.Fluorescence-activated cell sorting analysis, performed on isolatedhuman bone marrow and umbilical cord blood CD34-positive hematopoieticcells, demonstrated that a fraction of these cells are positive for Flt4(VEGFR-3). Thus, the VEGF-C effect on myelopoiesis may be exertedthrough this VEGFR-3-positive cell population and its receptors. In anycase, the foregoing data indicates a use for VEFG-C polypeptides toincrease granulocyte (and, in particular, neutrophil) counts in human ornon-human subjects, i.e., in order to assist the subject fightinfectious diseases. The exploitation of the myelopoietic activity ofVEGF-C polypeptides is contemplated both in vitro (i.e., in cellculture) and in vivo, as a sole myelopoietic agent and in combinationwith other effective agents (e.g., granulocyte colony stimulating factor(G-CSF)).

Additional studies of the myelopoietic effect of VEGF-C, using VEGF-Cmutants (e.g., VEGF-C ΔC₁₅₆ polypeptides, VEGF-C ΔNΔCHis, VEGF-CR226,227S) having altered VEGFR-2 binding affinities, will elucidatewhether this effect is mediated through VEGFR-2, VEGFR-3, or bothreceptors, for example. The results of such analysis will be useful indetermining which VEGF-C mutants have utility as myelopoietic agents andwhich have utility as agents for inhibiting myelopoiesis.

Example 37 Generation of Heterodimers Consisting of Members of the VEGFFamily of Growth Factors

Both naturally-occurring and recombinantly-produced heterodimers ofpolypeptides of the PDGF/VEGF family of growth factors have been shownto exist in nature and possess mitogenic activities. See, e.g., Cao etal., J. Biol. Chem., 271:3154-62 (1996); and DiSalvo, et al., J. Biol.Chem., 270:7717-7723 (1995). Heterodimers comprising a VEGF-Cpolypeptide may be generated essentially as described In Cao et al.(1996), using recombinantly produced VEGF-C polypeptides, such as theVEGF-C polypeptides described in the preceding examples. Briefly, arecombinantly produced VEGF-C polypeptide is mixed at an equimolar ratiowith another recombinantly produced polypeptide of interest, such as aVEGF, VEGF-B, PlGF, PDGFα, PDGFβ, or c-fos induced growth factorpolypeptide. (See, e.g., Cao et al. (1990); Collins et al., Nature,316:748-750 (1985) (PDGF-β, GenBank Acc. No. X02811); Claesson-Welsh etal., Proc. Natl. Acad. Sci. USA, 86(13):4917-4921 (1989) (PDGF-α,GenBank Acc. No. M22734); Claesson-Welsh et al., Mol. Cell. Biol.8:3476-3486 (1988) (PDGF-β, GenBank Acc. No. M21616); Olofsson et al.,Proc. Natl. Acad. Sci. (USA), 93:2576-2581 (1996) (VEGF-B, GenBank Acc.No. U48801); Maglione et al., Proc. Natl. Acad. Sci. (USA),88(20):9267-9271 (1996) (PlGF, GenBank Acc. No. X54936); Heldin et al.,Growth Factors, 8:245-252 (1993); Folkman, Nature Med., 1:27-31 (1995);Friesel et al., FASEB J, 9:919-25 (1995); Mustonen et al., J. Cell.Biol., 129:895-98 (1995); Orlandini, S., Proc. Natl. Acad. Sci. USA,93(21):11675-11680 (1996); and others cited elsewhere herein. The mixedpolypeptides are incubated in the presence of guanidine-HCl and DTT. Thethiol groups are then protected with S-sulfonation, and the protein isdialyzed overnight, initially against urea/glutathione-SH,glutathione-S—S-glutathione, and subsequently against 20 mM Tris-HCl.

In a preferred embodiment, a variety of differently processed VEGF-Cforms and VEGF-C variants and analogs, such as the ones described in thepreceding examples, are employed as the VEGF-C polypeptide used togenerate such heterodimers. Thereafter, the heterodimers are screened todetermine their binding affinity with respect to receptors of theVEGF/PDGF family (especially VEGFR-1, VEGFR-2, and VEGFR-3), and theirability to stimulate the receptors (e.g., assaying for dimer-stimulatedreceptor phosphorylation in cells expressing the receptor of interest ontheir surface). The binding assays may be competitive binding assayssuch as those described herein and in the art. In the initial bindingassays, recombinantly produced proteins comprising the extracellulardomains of receptors are employable, as described in preceding examplesfor VEGFR-2 and VEGFR-3. Heterodimers that bind and stimulate receptorsare useful as recombinant growth factor polypeptides. Heterodimers thatbind but do not stimulate receptors are useful as growth factorantagonists. Heterodimers that display agonistic or antagonisticactivities in the screening assays are further screened using, e.g.,endothelial cell migration assays, vascular permeability assays, and invivo assays. It will also be apparent from the preceding examples thatdimers comprising two VEGF-C polypeptides (i.e., dimers of identicalVEGF-C polypeptides as well as dimers of different VEGF-C polypeptides)are advantageously screened for agonistic and antagonistic activitiesusing the same assays.

In one preferred embodiment, VEGF-C ΔC₁₅₆ polypeptide is employed tomake the dimers. It is anticipated that agonists and antagonistscomprising a VEGF-C ΔC₁₅₆ polypeptide will have increased specificityfor stimulating and inhibiting VEGFR-3, without concomitant stimulationor inhibition of VEGFR-2.

In another preferred embodiment, VEGF-C polypeptides wherein theC-terminal proteolytic cleavage site has been altered to reduce oreliminate C-terminal processing (e.g. VEGF-C R226,227S) is employed tomake dimers for screening for inhibitory activity.

In yet another preferred embodiment, VEGF-C polypeptides comprisingamino-terminal fragments (e.g., the VEGF-C 15 kD form described herein)of VEGF-C are employed to make dimers.

It is further contemplated that inactivation of only one polypeptidechain in a dimer could be enough to generate an inhibitory molecule,which is demonstrated e.g., by the generation of PDGF inhibitory mutantas reported in Vassbotn, Mol. Cell. Biol., 13:4066-4076 (1993).Therefore, in one embodiment, inhibition is achieved by expression invivo of a polynucleotide (e.g., a cDNA construct) encoding theheterodimerization partner which is unable to bind (or bindsinefficiently) to the receptor, or by direct administration of thatmonomer in a pharmaceutical composition.

Example 38 Formation and Screening of Useful Recombinant VEGF/VEGF-CGenes and Polypeptides

Amino acid sequence comparison reveals that mature VEGF-C bearsstructural similarity to VEGF121 [Tischer et al., J. Biol. Chem.,266(18):11947-54 (1990], with certain noteworthy structural differences.For example, mature VEGF-C contains an unpaired cysteine (position 137of SEQ ID NO: 8) and is able to form non-covalently bonded polypeptidedimers. In one embodiment of the invention, a VEGF analog is createdwherein the unpaired cysteine residue from mature VEGF-C is introducedat an analogous position of VEGF (e.g., introduced at Leu₅₈ of the humanVEGF165 precursor (FIG. 2, Genbank Acc. No. M32977) to generate aVEGF^(+cys) mutant designated VEGF L58C). Such an alteration isintroduced into the VEGF165 coding sequence using site-directedmutagenesis procedures known in the art, such as the proceduresdescribed above in preceding examples to generate various VEGF-C mutantforms. This VEGF^(+cys) mutant is recombinantly expressed and isscreened (alone and as a heterodimer with other VEGF and VEGF-C forms)for VEGFR-2 and/or VEGFR-3 binding, stimulatory, and inhibitoryactivities, using in vitro and in vivo activity assays as describedelsewhere herein. To form another VEGF analog of the invention, aVEGF^(+cys) mutant is altered to remove a conserved cysteinecorresponding to cys₇₇ of the VEGF165 precursor. Elimination of thiscysteine from the VEGF L58C would result in a VEGF analog resemblingVEGF-CΔNΔCH is C156S. This VEGF analog is screened for itsVEGF-inhibitory activities with respect to VEGFR-2 and/or VEGFR-1 andfor VEGF-C like stimulatory or inhibitory activities.

Another noteworthy structural difference between VEGF and VEGF-C is theabsence in VEGF-C of several basic residues found in VEGF (e.g.,residues Arg₁₀₈, Lys₁₁₀ and His₁₁₂ in the VEGF165 precursor shown inFIG. 2) that have been implicated in VEGF receptor binding. See Keyt etal., J. Biol. Chem., 271(10):5638-46 (1996). In another embodiment ofthe invention, codons for basic residues (lys, arg, his) are substitutedinto the VEGF-C coding sequence at one or more analogous positions bysite-directed mutagenesis. For example, in a preferred embodiment,Glu₁₈₇, Thr₁₈₉, and Pro₁₉₁ in VEGF-C (SEQ ID NO: 8) are replaced withArg, Lys, and His residues, respectively. The resultant VEGF-C analogs(collectively termed “VEGF-C^(basic)” polypeptides) are recombinantlyexpressed and screened for VEGFR-1, VEGFR-2, and VEGFR-3 stimulatory andinhibitory activity. The foregoing VEGF and VEGF-C analogs that haveVEGF-like activity, VEGF-C-like activity, or that act as inhibitors ofVEGF or VEGF-C, are contemplated as additional aspects of the invention.Polynucleotides encoding the analogs also are intended as aspects of theinvention.

Example 39 Effects of VEGF-C on Growth and Differentiation of HumanCD34+ Progenitor Cells In Vitro

Human CD34+ progenitor cells (HPC, 10×10³) were isolated from bonemarrow or cord blood mononuclear cells using the MACS CD34 Progenitorcell Isolation Kit (Miltenyi Biotec, Bergish Gladbach, Germany),according to the instructions of the manufacturer and cultured in RPMI1640 medium supplemented with L-glutamine (2.5 mM), penicillin (125IE/ml), streptomycin (125 μg/ml) and pooled 10% umbilical cord blood(CB) plasma at 37° C. in a humidified atmosphere in the presence of 5%CO₂ for seven days, with or without VEGF-C and with or without one ofthe combinations of growth factors described below. Each experiment wasperformed in triplicate. After seven days, total cell number wasevaluated in each culture.

In a first set of experiments, VEGF-C was added, at concentrationsranging from 10 ng/ml to 1 μg/ml, to the cultures of CB CD34+ HPCs. Cellnumbers were evaluated at day 7 of culture. When added as a singlefactor, 100 ng/ml of VEGF-C was found support the survival andproliferation of only a few CD34+ HPCs under serum-free conditions. Withmedium alone, most of the cells died within a culture period of 7 days.However, there were consistently more cells in the cultures providedwith the VEGF-C.

A subsequent set of experiments investigated the co-stimulatory effectof VEGF-C in cultures either supplemented with recombinant human stemcell factor (rhSCF, 20 ng/ml PreproTech, Rocky Hill, N.Y.) alone or acombination of granulocyte macrophage colony stimulating factor(rhGM-CSF, 100 ng/ml, Sandoz, Basel, Switzerland) plus SCF. Addition ofVEGF-C to SCF-supplemented cultures resulted in a slight co-stimulatoryeffect on cell growth of CD34+ cells, and this effect was alreadyobservable at a VEGF-C concentration of 10 ng/ml. Addition of VEGF-C toGM-CSF-plus SCF-supplemented cultures clearly increased cell yieldsafter 7 days of culture, with an optimum VEGF-C concentration of 100ng/ml. Additional experiments were conducted to analyze theco-stimulatory effects of 100 ng/ml VEGF-C on total cell yields ofserum-free cultures of CB CD34+ HPC cells supplemented with eitherGM-CSF alone, IL-3 (rhIL-3, 100 U/ml, Behring AG, Marburg, Germany)alone; or a combination of GM-CSF plus IL-3. The results are shown belowin the following table:

Total cell number (E × 10⁻³) after a culture period of 7 days in RPMI +10% CBPL, + specified growth factors with (+) or without (−) VEGF-C.(Cell number at day 0 = 10) Growth experiment Factor(s) number −VEGF-C+VEGF-C GM-CSF 1 11 15 2 10 17 3 19 25 mean ± SE 13.3 ± 2.8  19.0 ± 3.1*IL-3 1 113 130 2 107 113 3 200 433 4 45 90 mean ± SE 116.2 ± 31.9 191.5± 80.9 GM-CSF + 1 150 160 IL-3 2 130 140 3 140 155 mean ± SE 140.0 ±5.7  151.7 ± 6.0* GM-CSF + 1 31 37 SCF 2 60 227 3 47 50 mean ± SE 46.0 ±8.3 104.7 ± 61.3 *mean ± SE; p = 0.02

As depicted in the table, VEGF-C led to a consistent enhancement of cellgrowth when added as a supplement to each growth factor or combinationof growth factors tested.

Effect of VEGF-C on Granulomonocytic Differentiation of Cd34+Progenitors

Using cells from the (7 day) plasma-supplemented cultures describedabove, immunofluorescence triple stainings were performed to analyze theexpression of the early granulomonocytic marker molecules lysozyme (LZ)and myeloperoxidase (MPO) as well as the lipopolysaccharide (LPS)receptor CD14. The table below depicts the percentages and numbers ofcells expressing MPO and/or LZ:

Percentages and numbers of cells expressing the markers MPO and LZ after7 days of culture with (+) or without (−) VEGF-C and specified growthfactors percent of cells positive numbers of cells positive for cellmarker for cell marker (E × 10⁻³) factor marker exp. no. −VEGF-C +VEGF-C−VEGF-C +VEGF-C GM-CSF MPO 1 57 69  6 11 2 45 53  5  9 3 18 24 10 13mean ± SE 40.0 ± 11.0 49.0 ± 13*  7.0 ± 1.5 11.0 ± 1.5* LZ 1 54 70  6 112 16 16  2  3 3 15 23  9 13 mean ± SE 28.0 ± 12.8 36.0 ± 16.7 5.7 ± 2.09.0 ± 3.0 IL-3 MPO 1 20 28 23 36 2 37 42 39 48 3  5  9 10 35 mean ± SE21.0 ± 9.0  26.0 ± 9.0  24.0 ± 8.3  39.7 ± 4.2  LZ 1 15 22 17 29 2  3  3 3  3 3  3  5  6 22 mean ± SE 7.0 ± 4.0 10.3 ± 5.8  8.7 ± 4.0 18.0 ±7.0  GM-CSF + IL-3 MPO 1 29 37 46 56 2 38 40 49 56 3  6 10  3  6 mean ±SE 24.0 ± 9.0  39.3 ± 16.6 32.7 ± 14.8 39.3 ± 16.6 LZ 1 18 20 29 30 2  2 3  3  3 3  1  2  1  2 mean ± SE 7.0 ± 5.5 8.3 ± 5.8 11.0 ± 9.0  12.0 ±9.0  GM-CSF + SCF MPO 1 50 51 15 19 2 16 21 10 48 mean ± SE 33.0 ± 17.036.0 ± 15.0 12.5 ± 2.5  33.5 ± 14.5 LZ 1 15 15  5  6 2  9 20  5 45 mean± SE 12.0 ± 3.0  18.0 ± 2.0  5.0 ± 0.0 25.5 ± 19.5Among the granulomonocytic markers tested, VEGF-C led to an increase inthe proportion of LZ+ cells under all culture conditions. In comparison,LZ+CD14+ cells, which represent differentiated monocytic cells only veryslightly increased upon addition of VEGF-C (data not shown).Co-stimulation of the cells with VEGF-C increased the expression of MPO,an early granulocytic marker molecule, only modestly, except incombination with both GM-CSF and IL-3, where the increase in theproportion of MPO+ cells was more pronounced.

VEGF-C Exerts Co-Stimulatory Effects in Combination with M-CSF

In another series of experiments, CD34+ cells were cultured in mediumsupplemented with 50 ng/ml M-CSF, with or without 100 ng/ml VEGF-C, forseven days. Culture of CD34+ cells in the presence of M-CSF leads to thegeneration of CD14+ monocytes within 7 days. After seven days, thecultures were analyzed to determine the percentages of CD14+ cells andmean fluorescence intensity. The results are summarized in the tablebelow:

Percentages of CD14⁺ cells and mean fluorescence intensity (MFI) ofcells cultured with M-CSF in the absence or in the presence of VEGF-CM-CSF alone M-CSF + VEGF-C exp no % CD14+ MFI % CD14+ MFI 1 37 20 47 402 42 44 54 74 3 32  6 36  7 mean ± SE 36.8 ± 2.9 23.3 ± 11.1 45.7 ± 5.240.3 ± 19.3As shown in the table, addition of VEGF-C to these cultures increasedboth the proportion of CD14+ cells (37% CD14+ cells vs. 46%) and thefluorescence intensity of CD14 expression (MFI 23.3 vs. 40.3). However,cell numbers did not increase upon addition of VEGF-C to M-CSFsupplemented cultures. Thus, VEGF-C had a small effect on thedifferentiation of monocytic cells, but not on their growth.

In the foregoing experiments the presence of VEGF-C was associated withenhanced numbers of cells in cultures of cord blood CD34+ cells. Underall conditions tested (GM-CSF, IL-3, GM-CSF+IL-3; GM-CSF+SCF),co-culture with VEGF-C led to an enhancement of proportions of myeloidcells. These results indicate an application for VEGF-C in thestimulation and/or differentiation of CD34+ progenitor cells in vitro orin vivo. Furthermore, the use of VEGF-C alone also slightly increasedthe number of surviving cells. The results thus indicate uses forcompositions comprising VEGF-C prepared in admixture with theaforementioned or other growth factors, such as VEGF-C, and unit doseformulations comprising VEGF-C packaged together with the aforementionedor other growth factors. Such compositions, unit dose formulations, andmethods of their use are intended as further aspects of the presentinvention.

Deposit of Biological Materials: Plasmid FLT4-L has been deposited withthe American Type Culture Collection (ATCC), 12301 Parklawn Dr.,Rockville Md. 20952 (USA), pursuant to the provisions of the BudapestTreaty, and has been assigned a deposit date of 24 Jul. 1995 and ATCCaccession number 97231.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those in the art. Accordingly, only such limitations as appearin the appended claims should be placed on the invention.

1. A method of inhibiting endothelial cell proliferation, comprisingadministering to a person a composition comprising an antibody thatspecifically binds with a soluble polypeptide ligand of Flt4 receptortyrosine kinase, wherein the soluble polypeptide ligand comprises apolypeptide encoded by the cDNA insert of plasmid pFlt4-L, having ATCCAccession No. 97231; wherein the polypeptide has a molecular weight ofabout 23 kD as assessed by SDS-PAGE under reducing conditions; andwherein binding of the antibody to the polypeptide ligand blocksactivation of Flt-4 receptor tyrosine kinase by the polypeptide ligand,wherein said antibody is administered in an amount effective to inhibitactivation of Flt4 by the ligand in the person, thereby inhibitingendothelial cell proliferation, wherein said antibody inhibits bindingbetween the polypeptide and Flt4.
 2. The method according to claim 1,wherein amino-terminal amino acids 2 through 18 of said polypeptidecorrespond to amino acids 2 through 18 set forth in SEQ ID NO:
 5. 3. Themethod according to claim 1, wherein the polypeptide binds andstimulates phosphorylation of VEGFR-2, and wherein the antibody inhibitsbinding between the polypeptide and VEGFR-2.
 4. The method according toclaim 1, wherein the antibody inhibits the polypeptide from stimulatingphosphorylation of Flt4.
 5. The method according to claim 4, wherein theantibody is a monoclonal antibody.
 6. The method according to claim 4,wherein the polypeptide binds and stimulates phosphorylation of VEGFR-2,and wherein the antibody inhibits the polypeptide from stimulatingphosphorylation of VEGFR-2.
 7. The method according to claim 6, whereinthe antibody is a monoclonal antibody.